Plants and cells transformed with a nucleic acid from Bacillus thuringiensis strain KB59A4-6 encoding a novel SUP toxin

ABSTRACT

The specification discloses a nucleic acid from  Bacillus thuringiensis  strain KB59A4-6 that encodes a novel pesticidal SUP toxin and plants and microbial cells transformed with the nucleic acid.

BACKGROUND OF THE INVENTION

Insects and other pests cost farmers billions of dollars annually incrop losses and in the expense of keeping these pests under control. Thelosses caused by insect pests in agricultural production environmentsinclude decrease in crop yield, reduced crop quality, and increasedharvesting costs.

Cultivation methods, such as crop rotation and the application of highnitrogen levels to stimulate the growth of an adventitious root system,has partially addressed problems caused by agricultural pests. Economicdemands on the utilization of farmland restrict the use of croprotation. In addition, overwintering traits of some insects aredisrupting crop rotations in some areas. Thus, chemical insecticides arerelied upon most heavily to guarantee the desired level of control.Insecticides are either banded onto or incorporated into the soil.

The use of chemical insecticides has several drawbacks. Continual use ofinsecticides has allowed resistant insects to evolve. Situations such asextremely high populations of larvae, heavy rains, and impropercalibration of insecticide application equipment can result in poorcontrol. The use of insecticides often raises environmental concernssuch as contamination of soil and of both surface and underground watersupplies. The public has also become concerned about the amount ofresidual, synthetic chemicals which might be found on food. Working withinsecticides may also pose hazards to the persons applying them.Therefore, synthetic chemical pesticides are being increasinglyscrutinized, and correctly so, for their potential toxic environmentalconsequences. Examples of widely used synthetic chemical pesticidesinclude the organochlorines, e.g., DDT, mirex, kepone, lindane, aldrin,chlordane, aldicarb, and dieldrin; the organophosphates, e.g.,chlorpyrifos, parathion, malathion, and diazinon; and carbamates.Stringent new restrictions on the use of pesticides and the eliminationof some effective pesticides from the market place could limiteconomical and effective options for controlling damaging and costlypests.

Because of the problems associated with the use of organic syntheticchemical pesticides, there exists a clear need to limit the use of theseagents and a need to identify alternative control agents. Thereplacement of synthetic chemical pesticides, or combination of theseagents with biological pesticides, could reduce the levels of toxicchemicals in the environment.

A biological pesticidal agent that is enjoying increasing popularity isthe soil microbe Bacillus thuringiensis (B.t.). The soil microbeBacillus thuringiensis (B.t.) is a Gram-positive, spore-formingbacterium. Most strains of B.t. do not exhibit pesticidal activity. SomeB.t. strains produce, and can be characterized by, parasporalcrystalline protein inclusions. These inclusions often appearmicroscopically as distinctively shaped crystals. Some B.t. proteins arehighly toxic to pests, such as insects, and are specific in their toxicactivity. Certain insecticidal B.t. proteins are associated with theinclusions. These “δ-endotoxins,” are different from exotoxins, whichhave a non-specific host range. Other species of Bacillus also producepesticidal proteins.

Certain Bacillus toxin genes have been isolated and sequenced, andrecombinant DNA-based products have been produced and approved for use.In addition, with the use of genetic engineering techniques, newapproaches for delivering these toxins to agricultural environments areunder development. These include the use of plants geneticallyengineered with toxin genes for insect resistance and the use ofstabilized intact microbial cells as toxin delivery vehicles. Thus,isolated Bacillus toxin genes are becoming commercially valuable.

Until the last fifteen years, commercial use of B.t. pesticides has beenlargely restricted to targeting a narrow range of lepidopteran(caterpillar) pests. Preparations of the spores and crystals of B.thuringiensis subsp.kurstaki have been used for many years as commercialinsecticides for lepidopteran pests. For example, B. thuringiensis var.kurstaki HD-1 produces a crystalline δ-endotoxin which is toxic to thelarvae of a number of lepidopteran insects.

In recent years, however, investigators have discovered B.t. pesticideswith specificities for a much broader range of pests. For example, otherspecies of B.t., namely israelensis and morrisoni (a.k.a. tenebrionis,a.k.a. B.t. M-7, a.k.a. B.t. san diego), have been used commercially tocontrol insects of the orders Diptera and Coleoptera, respectively.Bacillus thuringiensis var. tenebrionis has been reported to be activeagainst two beetles in the order Coleoptera (Colorado potato beetle,Leptinotarsa decemlineata, and Agelastica alni).

More recently, new subspecies of B.t. have been identified, and genesresponsible for active δ-endotoxin proteins have been isolated. Höfteand Whiteley classified B.t. crystal protein genes into four majorclasses (Höfte, H., H. R. Whiteley [1989] Microbiological Reviews52(2):242-255). The classes were CryI (Lepidoptera-specific), CryII(Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), andCryIV (Diptera-specific). The discovery of strains specifically toxic toother pests has been reported. For example, CryV and CryVI have beenproposed to designate a class of toxin genes that are nematode-specific.

The 1989 nomenclature and classification scheme of Höfte and Whiteleyfor crystal proteins was based on both the deduced amino acid sequenceand the host range of the toxin. That system was adapted to cover 14different types of toxin genes which were divided into five majorclasses. The number of sequenced Bacillus thuringiensis crystal proteingenes currently stands at more than 50. A revised nomenclature schemehas been proposed which is based solely on amino acid identity(Crickmore et al. [1996] Society for Invertebrate Pathology, 29th AnnualMeeting, IIIrd International Colloquium on Bacillus thuringiensis,University of Cordoba, Cordoba, Spain, Sep. 1-6, 1996, abstract). Themnemonic “cry” has been retained for all of the toxin genes except cytAand cytB, which remain a separate class. Roman numerals have beenexchanged for Arabic numerals in the primary rank, and the parenthesesin the tertiary rank have been removed. Many of the original names havebeen retained, with the noted exceptions, although a number have beenreclassified.

Many other B.t. genes have now been identified. WO 94/21795, WO96/10083, WO 98/44137, and Estruch, J. J. et al. (1996) PNAS93:5389-5394 describe Vip1A(a), Vip1A(b), Vip2A(a), Vip2A(b), Vip3A(a),and Vip3A(b) toxins obtained from Bacillus microbes. Those toxins arereported to be produced during vegetative cell growth and were thustermed vegetative insecticidal proteins (VIP). Activity of these toxinsagainst certain lepidopteran and certain coleopteran pests was reported.WO 98/18932 discloses new classes of pesticidal toxins.

Obstacles to the successful agricultural use of Bacillus toxins includethe development of resistance to B.t. toxins by insects. In addition,certain insects can be refractory to the effects of Bacillus toxins. Thelatter includes insects such as boll weevil and black cutworm as well asadult insects of most species which heretofore have demonstrated noapparent significant sensitivity to B.t. δ-endotoxins. While resistancemanagement strategies in B.t. transgene plant technology have become ofgreat interest, there remains a great need for developing additionalgenes that can be expressed in plants in order to effectively controlvarious insects.

The subject application provides new classes of toxins and genes, inaddition to those described in WO 98/18932, and which are distinct fromthose disclosed in WO 94/21795, WO 96/10083, WO 98/44137, and Estruch etal.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns materials and methods useful in thecontrol of non-mammalian pests and, particularly, plant pests. In oneembodiment, the subject invention provides novel Bacillus isolateshaving advantageous activity against non-mammalian pests. In a furtherembodiment, the subject invention provides new toxins useful for thecontrol of non-mammalian pests. In a preferred embodiment, these pestsare lepidopterans and/or coleopterans. The toxins of the subjectinvention include δ-endotoxins as well as soluble toxins which can beobtained from the supernatant of Bacillus cultures.

The subject invention further provides nucleotide sequences which encodethe toxins of the subject invention. The subject invention furtherprovides nucleotide sequences and methods useful in the identificationand characterization of genes which encode pesticidal toxins.

In one embodiment, the subject invention concerns unique nucleotidesequences which are useful as hybridization probes and/or primers in PCRtechniques. The primers produce characteristic gene fragments which canbe used in the identification, characterization, and/or isolation ofspecific toxin genes. The nucleotide sequences of the subject inventionencode toxins which are distinct from previously-described toxins.

In a specific embodiment, the subject invention provides new classes oftoxins having advantageous pesticidal activities. These classes oftoxins can be encoded by polynucleotide sequences which arecharacterized by their ability to hybridize with certain exemplifiedsequences and/or by their ability to be amplified by PCR using certainexemplified primers.

One aspect of the subject invention pertains to the identification andcharacterization of entirely new families of Bacillus toxins havingadvantageous pesticidal properties. The subject invention includes newclasses of genes and toxins referred to herein as MIS-7 and MIS-8. Genesand toxins of novel WAR- and SUP-classes are also disclosed. CertainMIS-1 and MIS-2 toxins and genes are also further characterized herein.

These families of toxins, and the genes which encode them, can becharacterized in terms of, for example, the size of the toxin or gene,the DNA or amino acid sequence, pesticidal activity, and/or antibodyreactivity. With regard to the genes encoding the novel toxin familiesof the subject invention, the current disclosure provides uniquehybridization probes and PCR primers which can be used to identify andcharacterize DNA within each of the exemplified families.

In one embodiment of the subject invention, Bacillus isolates can becultivated under conditions resulting in high multiplication of themicrobe. After treating the microbe to provide single-stranded genomicnucleic acid, the DNA can be contacted with the primers of the inventionand subjected to PCR amplification. Characteristic fragments oftoxin-encoding genes will be amplified by the procedure, thusidentifying the presence of the toxin-encoding gene(s).

A further aspect of the subject invention is the use of the disclosednucleotide sequences as probes to detect genes encoding Bacillus toxinswhich are active against pests.

Further aspects of the subject invention include the genes and isolatesidentified using the methods and nucleotide sequences disclosed herein.The genes thus identified encode toxins active against pests. Similarly,the isolates will have activity against these pests. In a preferredembodiment, these pests are lepidopteran or coleopteran pests.

In a preferred embodiment, the subject invention concerns plants cellstransformed with at least one polynucleotide sequence of the subjectinvention such that the transformed plant cells express pesticidaltoxins in tissues consumed by target pests. As described herein, thetoxins useful according to the subject invention may be chimeric toxinsproduced by combining portions of multiple toxins. In addition, mixturesand/or combinations of toxins can be used according to the subjectinvention.

Transformation of plants with the genetic constructs disclosed hereincan be accomplished using techniques well known to those skilled in theart and would typically involve modification of the gene to optimizeexpression of the toxin in plants.

Alternatively, the Bacillus isolates of the subject invention, orrecombinant microbes expressing the toxins described herein, can be usedto control pests. In this regard, the invention includes the treatmentof substantially intact Bacillus cells, and/or recombinant cellscontaining the expressed toxins of the invention, treated to prolong thepesticidal activity when the substantially intact cells are applied tothe environment of a target pest. The treated cell acts as a protectivecoating for the pesticidal toxin. The toxin becomes active uponingestion by a target insect.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 is a nucleotide sequence encoding a toxin from B.t. strainJavelin 1990.

SEQ ID NO. 2 is an amino acid sequence for the Javelin 1990 toxin.

SEQ ID NO. 3 is a forward primer used according to the subjectinvention.

SEQ ID NO. 4 is a reverse primer used according to the subjectinvention.

SEQ ID NO. 5 is a nucleotide sequence of a toxin gene from B.t. strainPS66D3

SEQ ID NO. 6 is an amino acid sequence from the 66D3 toxin.

SEQ ID NO. 7 is a nucleotide sequence of a MIS toxin gene from B.t.strain PS177C8.

SEQ ID NO. 8 is an amino acid sequence from the 177C8-MIS toxin.

SEQ ID NO. 9 is a nucleotide sequence of a toxin gene from B.t. strainPS177I8

SEQ ID NO. 10 is an amino acid sequence from the 177I8 toxin.

SEQ ID NO. 11 is a nucleotide sequence encoding a 177C8-WAR toxin genefrom B.t. strain PS177C8.

SEQ ID NO. 12 is an amino acid sequence of a 177C8-WAR toxin from B.t.strain PS177C8.

SEQ ID NOS. 13-21 are primers used according to the subject invention.

SEQ ID NO. 22 is the reverse complement of the primer of SEQ ID NO. 14.

SEQ ID NO. 23 is the reverse complement of the primer of SEQ ID NO. 15.

SEQ ID NO. 24 is the reverse complement of the primer of SEQ ID NO. 17.

SEQ ID NO. 25 is the reverse complement of the primer of SEQ ID NO. 18.

SEQ ID NO. 26 is the reverse complement of the primer of SEQ ID NO. 19.

SEQ ID NO. 27 is the reverse complement of the primer of SEQ ID NO. 20.

SEQ ID NO. 28 is the reverse complement of the primer of SEQ ID NO. 21.

SEQ ID NO. 29 is a MIS-7 forward primer.

SEQ ID NO. 30 is a MIS-7 reverse primer.

SEQ ID NO. 31 is a MIS-8 forward primer.

SEQ ID NO. 32 is a MIS-8 reverse primer.

SEQ ID NO. 33 is a nucleotide sequence of a MIS-7 toxin gene designated157C1-A from B.t. strain PS157C1.

SEQ ID NO. 34 is an amino acid sequence of a MIS-7 toxin designated157C1-A from B.t. strain PS157C1.

SEQ ID NO. 35 is a nucleotide sequence of a MIS-7 toxin gene from B.t.strain PS201Z.

SEQ ID NO. 36 is a nucleotide sequence of a MIS-8 toxin gene from B.t.strain PS31F2.

SEQ ID NO. 37 is a nucleotide sequence of a MIS-8 toxin gene from B.t.strain PS185Y2.

SEQ ID NO. 38 is a nucleotide sequence of a MIS-1 toxin gene from B.t.strain PS33F1.

SEQ ID NO. 39 is a MIS primer for use according to the subjectinvention.

SEQ ID NO. 40 is a MIS primer for use according to the subjectinvention.

SEQ ID NO. 41 is a WAR primer for use according to the subjectinvention.

SEQ ID NO. 42 is a WAR primer for use according to the subjectinvention.

SEQ ID NO. 43 is a partial nucleotide sequence for a MIS-7 gene fromPS205C.

SEQ ID NO. 44 is a partial amino acid sequence for a MIS-7 toxin fromPS205C.

SEQ ID NO. 45 is a partial nucleotide sequence for a WAR gene fromPS205C.

SEQ ID NO. 46 is a partial amino acid sequence for a WAR toxin fromPS205C.

SEQ ID NO. 47 is a nucleotide sequence for a MIS-8 gene from PS31F2.

SEQ ID NO. 48 is an amino acid sequence for a MIS-8 toxin from PS31F2.

SEQ ID NO. 49 is a nucleotide sequence for a WAR gene from PS31F2.

SEQ ID NO. 50 is an amino acid sequence for a WAR toxin from PS31F2.

SEQ ID NO. 51 is a SUP primer for use according to the subjectinvention.

SEQ ID NO. 52 is a SUP primer for use according to the subjectinvention.

SEQ ID NO. 53 is a nucleotide sequence for a SUP gene from KB59A4-6.

SEQ ID NO. 54 is an amino acid sequence for a SUP toxin from KB59A4-6.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns materials and methods for the control ofnon-mammalian pests. In specific embodiments, the subject inventionpertains to new Bacillus thuringiensis isolates and toxins which haveactivity against lepidopterans and/or coleopterans. The subjectinvention further concerns novel genes which encode pesticidal toxinsand novel methods for identifying and characterizing Bacillus geneswhich encode toxins with useful properties. The subject inventionconcerns not only the polynucleotide sequences which encode thesetoxins, but also the use of these polynucleotide sequences to producerecombinant hosts which express the toxins. The proteins of the subjectinvention are distinct from protein toxins which have previously beenisolated from Bacillus thuringiensis.

B.t. isolates useful according to the subject invention have beendeposited in the permanent collection of the Agricultural ResearchService Patent Culture Collection (NRRL), Northern Regional ResearchCenter, 1815 North University Street, Peoria, Ill. 61604, USA. Theculture repository numbers of the B.t. strains are as follows:

TABLE 1 Culture Repository No. Deposit Date Patent No. B.t. PS157C1(MT104) NRRL B-18240 Jul. 17, 1987 5,262,159 B.t. PS31F2 NRRL B-21876Oct. 24, 1997 B.t. PS66D3 NRRL B-21858 Oct. 24, 1997 B.t. PS177C8a NRRLB-21867 Oct. 24, 1997 B.t. PS177I8 NRRL B-21868 Oct. 24, 1997 KB53A49-4NRRL B-21879 Oct. 24, 1997 KB68B46-2 NRRL B-21877 Oct. 24, 1997KB68B51-2 NRRL B-21880 Oct. 24, 1997 KB68B55-2 NRRL B-21878 Oct. 24,1997 PS33F1 NRRL B-21977 Apr. 24, 1998 PS71G4 NRRL B-21978 Apr. 24, 1998PS86D1 NRRL B-21979 Apr. 24, 1998 5185V2 NRRL B-21980 Apr. 24, 1998S191A21 NRRL B-21981 Apr. 24, 1998 P5201Z NRRL B-21982 Apr. 24, 1998PS205A3 NRRL B-21983 Apr. 24, 1998 PS205C NRRL B-21984 Apr. 24, 1998PS234E1 NRRL B-21985 Apr. 24, 1998 P5248N10 NRRL B-21986 Apr. 24, 1998KB63B19-13 NRRL B-21990 Apr. 29, 1998 KB63B19-7 NRRL B-21989 Apr. 29,1998 KB68B62-7 NRRL B-21991 Apr. 29, 1998 KB68B63-2 NRRL B-21992 Apr.29, 1998 KB69A125-1 NRRL B-21993 Apr. 29, 1998 KB69A125-3 NRRL B-21994Apr. 29, 1998 KB69A125-5 NRRL B-21995 Apr. 29, 1998 KB69A127-7 NRRLB-21996 Apr. 29, 1998 KB69A132-1 NRRL B-21997 Apr. 29, 1998 KB69B2-1NRRL B-21998 Apr. 29, 1998 KB70B5-3 NRRL B-21999 Apr. 29, 1998KB71A125-15 NRRL B-30001 Apr. 29, 1998 KB71A35-6 NRRL B-30000 Apr. 29,1998 KB71A72-1 NRRL B-21987 Apr. 29, 1998 KB71A134-2 NRRL B-21988 Apr.29, 1998 P5185Y2 NRRL B-30121 May 4, 1999 KBS9A4-6 NRRL B-30173 Aug. 5,1999 MR992 NRRL B-30124 May 4, 1999 MR983 NRRL B-30123 May 4, 1999 MR993NRRL B-30125 May 4, 1999 MR951 NRRL B-30122 May 4, 1999

Cultures which have been deposited for the purposes of this patentapplication were deposited under conditions that assure that access tothe cultures is available during the pendency of this patent applicationto one determined by the Commissioner of Patents and Trademarks to beentitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposits willbe available as required by foreign patent laws in countries whereincounterparts of the subject application, or its progeny, are filed.However, it should be understood that the availability of a deposit doesnot constitute a license to practice the subject invention in derogationof patent rights granted by governmental action.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of the deposit, and in any case, for a period of at least thirty(30) years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the culture(s). The depositoracknowledges the duty to replace the deposit(s) should the depository beunable to furnish a sample when requested, due to the condition of adeposit. All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

Many of the strains useful according to the subject invention arereadily available by virtue of the issuance of patents disclosing thesestrains or by their deposit in public collections or by their inclusionin commercial products. For example, the B.t. strain used in thecommercial product, Javelin, and the HD isolates are all publiclyavailable.

Mutants of the isolates referred to herein can be made by procedureswell known in the art. For example, an asporogenous mutant can beobtained through ethylmethane sulfonate (EMS) mutagenesis of an isolate.The mutants can be made using ultraviolet light and nitrosoguanidine byprocedures well known in the art.

In one embodiment, the subject invention concerns materials and methodsincluding nucleotide primers and probes for isolating, characterizing,and identifying Bacillus genes encoding protein toxins which are activeagainst non-mammalian pests. The nucleotide sequences described hereincan also be used to identify new pesticidal Bacillus isolates. Theinvention further concerns the genes, isolates, and toxins identifiedusing the methods and materials disclosed herein.

The new toxins and polynucleotide sequences provided here are definedaccording to several parameters. One characteristic of the toxinsdescribed herein is pesticidal activity. In a specific embodiment, thesetoxins have activity against coleopteran and/or lepidopteran pests. Thetoxins and genes of the subject invention can be further defined bytheir amino acid and nucleotide sequences. The sequences of themolecules can be defined in terms of homology to certain exemplifiedsequences as well as in terms of the ability to hybridize with, or beamplified by, certain exemplified probes and primers. The toxinsprovided herein can also be identified based on their immunoreactivitywith certain antibodies.

An important aspect of the subject invention is the identification andcharacterization of new families of Bacillus toxins, and genes whichencode these toxins. These families have been designated MIS-7 andMIS-8. New WAR- and SUP-type toxin families are also disclosed herein.Toxins within these families, as well as genes encoding toxins withinthese families, can readily be identified as described herein by, forexample, size, amino acid or DNA sequence, and antibody reactivity.Amino acid and DNA sequence characteristics include homology withexemplified sequences, ability to hybridize with DNA probes, and abilityto be amplified with specific primers.

A gene and toxin (which are obtainable from PS33F1) of the MIS-1 familyand a gene and toxin (which are obtainable from PS66D3) of the MIS-2family are also further characterized herein.

A novel family of toxins identified herein is the MIS-7 family. Thisfamily includes toxins which can be obtained from B.t. isolates PS157C1,PS205C, and PS201Z. The subject invention further provides probes andprimers for identification of the MIS-7 genes and toxins.

A further, novel family of toxins identified herein is the MIS-8 family.This family includes toxins which can be obtained from B.t. isolatesPS31F2 and PS185Y2. The subject invention further provides probes andprimers for identification of the MIS-8 genes and toxins.

In a preferred embodiment, the genes of the MIS family encode toxinshaving a molecular weight of about 70 to about 100 kDa and, mostpreferably, the toxins have a size of about 80 kDa. Typically, thesetoxins are soluble and can be obtained from the supernatant of Bacilluscultures as described herein. These toxins have toxicity againstnon-mammalian pests. In a preferred embodiment, these toxins haveactivity against coleopteran pests. The MIS proteins are further usefuldue to their ability to form pores in cells. These proteins can be usedwith second entities including, for example, other proteins. When usedwith a second entity, the MIS protein will facilitate entry of thesecond agent into a target cell. In a preferred embodiment, the MISprotein interacts with MIS receptors in a target cell and causes poreformation in the target cell. The second entity may be a toxin oranother molecule whose entry into the cell is desired.

The subject invention further concerns a family of toxins designatedWAR-type toxins. The WAR toxins typically have a size of about 30-50 kDaand, most typically, have a size of about 40 kDa. Typically, thesetoxins are soluble and can be obtained from the supernatant of Bacilluscultures as described herein. The WAR toxins can be identified withprimers described herein as well as with antibodies.

An additional family of toxins provided according to the subjectinvention are the toxins designated SUP-type toxins. Typically, thesetoxins are soluble and can be obtained from the supernatant of Bacilluscultures as described herein. In a preferred embodiment, the SUP toxinsare active against lepidopteran pests. The SUP toxins typically have asize of about 70-100 kDa and, preferably, about 80 kDa. The SUP familyis exemplified herein by toxins from isolate KB59A4-6. The subjectinvention provides probes and primers useful for the identification oftoxins and genes in the SUP family.

The subject invention also provides additional Bacillus toxins andgenes, including additional MIS, WAR, and SUP toxins and genes.

Toxins in the MIS, WAR, and SUP families are all soluble and can beobtained as described herein from the supernatant of Bacillus cultures.These toxins can be used alone or in combination with other toxins tocontrol pests. For example, toxins from the MIS families may be used inconjunction with WAR-type toxins to achieve control of pests,particularly coleopteran pests. These toxins may be used, for example,with δ-endotoxins which are obtained from Bacillus isolates.

Table 2 provides a summary of the novel families of toxins and genes ofthe subject invention. Certain MIS families are specifically exemplifiedherein by toxins which can be obtained from particular B.t. isolates asshown in Table 2. Genes encoding toxins in each of these families can beidentified by a variety of highly specific parameters, including theability to hybridize with the particular probes set forth in Table 2.Sequence identity in excess of about 80% with the probes set forth inTable 2 can also be used to identify the genes of the various families.Also exemplified are particular primer pairs which can be used toamplify the genes of the subject invention. A portion of a gene withinthe indicated families would typically be amplifiable with at least oneof the enumerated primer pairs. In a preferred embodiment, the amplifiedportion would be of approximately the indicated fragment size. Primersshown in Table 2 consist of polynucleotide sequences which encodepeptides as shown in the sequence listing attached hereto. Additionalprimers and probes can readily be constructed by those skilled in theart such that alternate polynucleotide sequences encoding the same aminoacid sequences can be used to identify and/or characterize additionalgenes encoding pesticidal toxins. In a preferred embodiment, theseadditional toxins, and their genes, could be obtained from Bacillusisolates.

TABLE 2 Probes Fragment (SEQ ID Primer Pairs size Family Isolates NO.)(SEQ ID NOS.) (nt) MIS-1 PS33F1 37  13 and 22  69 13 and 23 506 14 and23 458 MIS-2 PS66D3 5 16 and 24 160 16 and 25 239 16 and 26 400 16 and27 509 16 and 28 703 17 and 25 102 17 and 26 263 17 and 27 372 17 and 28566 18 and 26 191 18 and 27 300 18 and 28 494 19 and 27 131 19 and 28325 20 and 28 213 MIS-7 PS205C, PS157C1 33, 35 29 and 30 598 (157C1-A),PS201Z MIS-8 PS31F2, PS185Y2 36, 37 31 and 32 585 SUP KB59A4-6 1 51 and52

Furthermore, chimeric toxins may be used according to the subjectinvention. Methods have been developed for making useful chimeric toxinsby combining portions of B.t. proteins. The portions which are combinedneed not, themselves, be pesticidal so long as the combination ofportions creates a chimeric protein which is pesticidal. This can bedone using restriction enzymes, as described in, for example, EuropeanPatent 0 228 838; Ge, A. Z., N. L. Shivarova, D. H. Dean (1989) Proc.Natl. Acad. Sci. USA 86:4037-4041; Ge, A. Z., D. Rivers, R. Milne, D. H.Dean (1991) J. Biol. Chem. 266:17954-17958; Schnepf, H. E., K. Tomczak,J. P. Ortega, H. R. Whiteley (1990) J. Biol. Chem. 265:20923-20930;Honee, G., D. Convents, J. Van Rie, S. Jansens, M. Peferoen, B. Visser(1991) Mol. Microbiol. 5:2799-2806. Alternatively, recombination usingcellular recombination mechanisms can be used to achieve similarresults. See, for example, Caramori, T., A. M. Albertini, A. Galizzi(1991) Gene 98:37-44; Widner, W. R., H. R. Whiteley (1990) J. Bacteriol.172:2826-2832; Bosch, D., B. Schipper, H. van der Kliej, R. A. de Maagd,W. J. Stickema (1994) Biotechnology 12:915-918. A number of othermethods are known in the art by which such chimeric DNAs can be made.The subject invention is meant to include chimeric proteins that utilizethe novel sequences identified in the subject application.

With the teachings provided herein, one skilled in the art could readilyproduce and use the various toxins and polynucleotide sequencesdescribed herein.

Genes and toxins. The genes and toxins useful according to the subjectinvention include not only the full length sequences but also fragmentsof these sequences, variants, mutants, and fusion proteins which retainthe characteristic pesticidal activity of the toxins specificallyexemplified herein. Chimeric genes and toxins, produced by combiningportions from more than one Bacillus toxin or gene, may also be utilizedaccording to the teachings of the subject invention. As used herein, theterms “variants” or “variations” of genes refer to nucleotide sequenceswhich encode the same toxins or which encode equivalent toxins havingpesticidal activity. As used herein, the term “equivalent toxins” refersto toxins having the same or essentially the same biological activityagainst the target pests as the exemplified toxins. For example, U.S.Pat. No. 5,605,793 describes methods for generating additional moleculardiversity by using DNA reassembly after random fragmentation.

It is apparent to a person skilled in this art that genes encodingactive toxins can be identified and obtained through several means. Thespecific genes exemplified herein may be obtained from the isolatesdeposited at a culture depository as described above. These genes, orportions or variants thereof, may also be constructed synthetically, forexample, by use of a gene synthesizer. Variations of genes may bereadily constructed using standard techniques for making pointmutations. Also, fragments of these genes can be made using commerciallyavailable exonucleases or endonucleases according to standardprocedures. For example, enzymes such as Bal31 or site-directedmutagenesis can be used to systematically cut off nucleotides from theends of these genes. Also, genes which encode active fragments may beobtained using a variety of restriction enzymes. Proteases may be usedto directly obtain active fragments of these toxins.

Equivalent toxins and/or genes encoding these equivalent toxins can bederived from Bacillus isolates and/or DNA libraries using the teachingsprovided herein. There are a number of methods for obtaining thepesticidal toxins of the instant invention. For example, antibodies tothe pesticidal toxins disclosed and claimed herein can be used toidentify and isolate toxins from a mixture of proteins. Specifically,antibodies may be raised to the portions of the toxins which are mostconstant and most distinct from other Bacillus toxins. These antibodiescan then be used to specifically identify equivalent toxins with thecharacteristic activity by immunoprecipitation, enzyme linkedimmunosorbent assay (ELISA), or Western blotting. Antibodies to thetoxins disclosed herein, or to equivalent toxins, or fragments of thesetoxins, can readily be prepared using standard procedures in this art.The genes which encode these toxins can then be obtained from themicroorganism.

Fragments and equivalents which retain the pesticidal activity of theexemplified toxins are within the scope of the subject invention. Also,because of the redundancy of the genetic code, a variety of differentDNA sequences can encode the amino acid sequences disclosed herein. Itis well within the skill of a person trained in the art to create thesealternative DNA sequences encoding the same, or essentially the same,toxins. These variant DNA sequences are within the scope of the subjectinvention. As used herein, reference to “essentially the same” sequencerefers to sequences which have amino acid substitutions, deletions,additions, or insertions which do not materially affect pesticidalactivity. Fragments retaining pesticidal activity are also included inthis definition.

A further method for identifying the toxins and genes of the subjectinvention is through the use of oligonucleotide probes. These probes aredetectable nucleotide sequences. Probes provide a rapid method foridentifying toxin-encoding genes of the subject invention. Thenucleotide segments which are used as probes according to the inventioncan be synthesized using a DNA synthesizer and standard procedures.

Certain toxins of the subject invention have been specificallyexemplified herein. Since these toxins are merely exemplary of thetoxins of the subject invention, it should be readily apparent that thesubject invention comprises variant or equivalent toxins (and nucleotidesequences coding for equivalent toxins) having the same or similarpesticidal activity of the exemplified toxin. Equivalent toxins willhave amino acid homology with an exemplified toxin. This amino acididentity will typically be greater than 60%, preferably be greater than75%, more preferably greater than 80%, more preferably greater than 90%,and can be greater than 95%. These identities are as determined usingstandard alignment techniques. The amino acid homology will be highestin critical regions of the toxin which account for biological activityor are involved in the determination of three-dimensional configurationwhich ultimately is responsible for the biological activity. In thisregard, certain amino acid substitutions are acceptable and can beexpected if these substitutions are in regions which are not critical toactivity or are conservative amino acid substitutions which do notaffect the three-dimensional configuration of the molecule. For example,amino acids may be placed in the following classes: non-polar, unchargedpolar, basic, and acidic. Conservative substitutions whereby an aminoacid of one class is replaced with another amino acid of the same typefall within the scope of the subject invention so long as thesubstitution does not materially alter the biological activity of thecompound. Table 3 provides a listing of examples of amino acidsbelonging to each class.

TABLE 3 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not significantlydetract from the biological activity of the toxin.

The δ-endotoxins of the subject invention can also be characterized interms of the shape and location of toxin inclusions, which are describedabove.

As used herein, reference to “isolated” polynucleotides and/or“purified” toxins refers to these molecules when they are not associatedwith the other molecules with which they would be found in nature. Thus,reference to “isolated and purified” signifies the involvement of the“hand of man” as described herein. Chimeric toxins and genes alsoinvolve the “hand of man.”

Recombinant hosts. The toxin-encoding genes of the subject invention canbe introduced into a wide variety of microbial or plant hosts.Expression of the toxin gene results, directly or indirectly, in theproduction and maintenance of the pesticide. With suitable microbialhosts, e.g., Pseudomonas, the microbes can be applied to the situs ofthe pest, where they will proliferate and be ingested. The result is acontrol of the pest. Alternatively, the microbe hosting the toxin genecan be killed and treated under conditions that prolong the activity ofthe toxin and stabilize the cell. The treated cell, which retains thetoxic activity, then can be applied to the environment of the targetpest.

Where the Bacillus toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, it is essential that certain host microbes be used. Microorganismhosts are selected which are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest. These microorganisms are selected so as to becapable of successfully competing in the particular environment (cropand other insect habitats) with the wild-type microorganisms, providefor stable maintenance and expression of the gene expressing thepolypeptide pesticide, and, desirably, provide for improved protectionof the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodtorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. dorus,Kluyveromyces veronae, and Aureobasidium pollulans. Of particularinterest are the pigmented microorganisms.

A wide variety of ways are available for introducing a Bacillus geneencoding a toxin into a microorganism host under conditions which allowfor stable maintenance and expression of the gene. These methods arewell known to those skilled in the art and are described, for example,in U.S. Pat. No. 5,135,867, which is incorporated herein by reference.

Synthetic genes which are functionally equivalent to the toxins of thesubject invention can also be used to transform hosts. Methods for theproduction of synthetic genes can be found in, for example, U.S. Pat.No. 5,380,831.

Treatment of cells. As mentioned above, Bacillus or recombinant cellsexpressing a Bacillus toxin can be treated to prolong the toxin activityand stabilize the cell. The pesticide microcapsule that is formedcomprises the Bacillus toxin within a cellular structure that has beenstabilized and will protect the toxin when the microcapsule is appliedto the environment of the target pest. Suitable host cells may includeeither prokaryotes or eukaryotes. As hosts, of particular interest willbe the prokaryotes and the lower eukaryotes, such as fungi. The cellwill usually be intact and be substantially in the proliferative formwhen treated, rather than in a spore form.

Treatment of the microbial cell, e.g., a microbe containing the Bacillustoxin gene, can be by chemical or physical means, or by a combination ofchemical and/or physical means, so long as the technique does notdeleteriously affect the properties of the toxin, nor diminish thecellular capability of protecting the toxin. Methods for treatment ofmicrobial cells are disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462,which are incorporated herein by reference.

Methods and formulations for control of pests. Control of pests usingthe isolates, toxins, and genes of the subject invention can beaccomplished by a variety of methods known to those skilled in the art.These methods include, for example, the application of Bacillus isolatesto the pests (or their location), the application of recombinantmicrobes to the pests (or their locations), and the transformation ofplants with genes which encode the pesticidal toxins of the subjectinvention. Transformations can be made by those skilled in the art usingstandard techniques. Materials necessary for these transformations aredisclosed herein or are otherwise readily available to the skilledartisan.

Formulated bait granules containing an attractant and the toxins of theBacillus isolates, or recombinant microbes comprising the genesobtainable from the Bacillus isolates disclosed herein, can be appliedto the soil. Formulated product can also be applied as a seed-coating orroot treatment or total plant treatment at later stages of the cropcycle. Plant and soil treatments of Bacillus cells may be employed aswettable powders, granules or dusts, by mixing with various inertmaterials, such as inorganic minerals (phyllosilicates, carbonates,sulfates, phosphates, and the like) or botanical materials (powderedcorncobs, rice hulls, walnut shells, and the like). The formulations mayinclude spreader-sticker adjuvants, stabilizing agents, other pesticidaladditives, or surfactants. Liquid formulations may be aqueous-based ornon-aqueous and employed as foams, gels, suspensions, emulsifiableconcentrates, or the like. The ingredients may include rheologicalagents, surfactants, emulsifiers, dispersants, or polymers.

As would be appreciated by a person skilled in the art, the pesticidalconcentration will vary widely depending upon the nature of theparticular formulation, particularly whether it is a concentrate or tobe used directly. The pesticide will be present in at least 1% by weightand may be 100% by weight. The dry formulations will have from about1-95% by weight of the pesticide while the liquid formulations willgenerally be from about 1-60% by weight of the solids in the liquidphase. The formulations that contain cells will generally have fromabout 102 to about 104 cells/mg. These formulations will be administeredat about 50 mg (liquid or dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the pest, e.g.,soil and foliage, by spraying, dusting, sprinkling, or the like.

Polynucleotide probes. It is well known that DNA possesses a fundamentalproperty called base complementarity. In nature, DNA ordinarily existsin the form of pairs of anti-parallel strands, the bases on each strandprojecting from that strand toward the opposite strand. The base adenine(A) on one strand will always be opposed to the base thymine (T) on theother strand, and the base guanine (G) will be opposed to the basecytosine (C). The bases are held in apposition by their ability tohydrogen bond in this specific way. Though each individual bond isrelatively weak, the net effect of many adjacent hydrogen bonded bases,together with base stacking effects, is a stable joining of the twocomplementary strands. These bonds can be broken by treatments such ashigh pH or high temperature, and these conditions result in thedissociation, or “denaturation,” of the two strands. If the DNA is thenplaced in conditions which make hydrogen bonding of the basesthermodynamically favorable, the DNA strands will anneal, or“hybridize,” and reform the original double stranded DNA. If carried outunder appropriate conditions, this hybridization can be highly specific.That is, only strands with a high degree of base complementarity will beable to form stable double stranded structures. The relationship of thespecificity of hybridization to reaction conditions is well known. Thus,hybridization may be used to test whether two pieces of DNA arecomplementary in their base sequences. It is this hybridizationmechanism which facilitates the use of probes of the subject inventionto readily detect and characterize DNA sequences of interest.

The probes may be RNA, DNA, or PNA (peptide nucleic acid). The probewill normally have at least about 10 bases, more usually at least about17 bases, and may have up to about 100 bases or more. Longer probes canreadily be utilized, and such probes can be, for example, severalkilobases in length. The probe sequence is designed to be at leastsubstantially complementary to a portion of a gene encoding a toxin ofinterest. The probe need not have perfect complementarity to thesequence to which it hybridizes. The probes may be labelled utilizingtechniques which are well known to those skilled in this art.

One approach for the use of the subject invention as probes entailsfirst identifying by Southern blot analysis of a gene bank of theBacillus isolate all DNA segments homologous with the disclosednucleotide sequences. Thus, it is possible, without the aid ofbiological analysis, to know in advance the probable activity of manynew Bacillus isolates, and of the individual gene products expressed bya given Bacillus isolate. Such a probe analysis provides a rapid methodfor identifying potentially commercially valuable insecticidal toxingenes within the multifarious subspecies of B.t.

One hybridization procedure useful according to the subject inventiontypically includes the initial steps of isolating the DNA sample ofinterest and purifying it chemically. Either lysed bacteria or totalfractionated nucleic acid isolated from bacteria can be used. Cells canbe treated using known techniques to liberate their DNA (and/or RNA).The DNA sample can be cut into pieces with an appropriate restrictionenzyme. The pieces can be separated by size through electrophoresis in agel, usually agarose or acrylamide. The pieces of interest can betransferred to an immobilizing membrane.

The particular hybridization technique is not essential to the subjectinvention. As improvements are made in hybridization techniques, theycan be readily applied.

The probe and sample can then be combined in a hybridization buffersolution and held at an appropriate temperature until annealing occurs.Thereafter, the membrane is washed free of extraneous materials, leavingthe sample and bound probe molecules typically detected and quantifiedby autoradiography and/or liquid scintillation counting. As is wellknown in the art, if the probe molecule and nucleic acid samplehybridize by forming a strong non-covalent bond between the twomolecules, it can be reasonably assumed that the probe and sample areessentially identical. The probe's detectable label provides a means fordetermining in a known manner whether hybridization has occurred.

In the use of the nucleotide segments as probes, the particular probe islabeled with any suitable label known to those skilled in the art,including radioactive and non-radioactive labels. Typical radioactivelabels include ³²P, ³⁵S, or the like. Non-radioactive labels include,for example, ligands such as biotin or thyroxine, as well as enzymessuch as hydrolases or perixodases, or the various chemilurninescers suchas luciferin, or fluorescent compounds like fluorescein and itsderivatives. The probes may be made inherently fluorescent as describedin International Application No. WO 93/16094.

Various degrees of stringency of hybridization can be employed. The moresevere the conditions, the greater the complementarity that is requiredfor duplex formation. Severity can be controlled by temperature, probeconcentration, probe length, ionic strength, time, and the like.Preferably, hybridization is conducted under moderate to high stringencyconditions by techniques well known in the art, as described, forexample, in Keller, G. H., M. M. Manak (1987) DNA Probes, StocktonPress, New York, N.Y., pp. 169-170.

As used herein “moderate to high stringency” conditions forhybridization refers to conditions which achieve the same, or about thesame, degree of specificity of hybridization as the conditions employedby the current applicants. Examples of moderate and high stringencyconditions are provided herein. Specifically, hybridization ofimmobilized DNA on Southern blots with 32P-labeled gene-specific probeswas performed by standard methods (Maniatis et al.). In general,hybridization and subsequent washes were carried out under moderate tohigh stringency conditions that allowed for detection of targetsequences with homology to the exemplified toxin genes. Fordouble-stranded DNA gene probes, hybridization was carried out overnightat 20-25° C. below the melting temperature (Tm) of the DNA hybrid in6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Themelting temperature is described by the following formula (Beltz, G. A.,K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, and F. C. Kafatos [1983]Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [eds.] AcademicPress, New York 100:266-285).

Tm=81.5° C.+16.6 Log[Na+]+0.41(%G+C)−0.61(%formamide)−600/length ofduplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (lowstringency wash).

(2) Once at Tm−20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderatestringency wash).

For oligonucleotide probes, hybridization was carried out overnight at10-20° C. below the melting temperature (Tm) of the hybrid in 6×SSPE,5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm foroligonucleotide probes was determined by the following formula:

Tm (° C.)=2(number T/A base pairs)+4(number G/C base pairs) (Suggs, S.V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura, and R. B.Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, D. D.Brown [ed.], Academic Press, New York, 23:683-693).

Washes were typically carried out as follows:

(1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS (lowstringency wash).

(2) Once at the hybridization temperature for 15 minutes in 1×SSPE, 0.1%SDS (moderate stringency wash).

In general, salt and/or temperature can be altered to change stringency.With a labeled DNA fragment>70 or so bases in length, the followingconditions can be used:

Low: 1 or 2×SSPE, room temperature

Low: 1 or 2×SSPE, 42° C.

Moderate: 0.2× or 1×SSPE, 65° C.

High: 0.1×SSPE, 65° C.

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid, and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probe sequences ofthe subject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions, and deletions can be produced in a givenpolynucleotide sequence in many ways, and these methods are known to anordinarily skilled artisan. Other methods may become known in thefuture.

Thus, mutational, insertional, and deletional variants of the disclosednucleotide sequences can be readily prepared by methods which are wellknown to those skilled in the art. These variants can be used in thesame manner as the exemplified primer sequences so long as the variantshave substantial sequence homology with the original sequence. As usedherein, substantial sequence homology refers to homology which issufficient to enable the variant probe to function in the same capacityas the original probe. Preferably, this homology is greater than 50%;more preferably, this homology is greater than 75%; and most preferably,this homology is greater than 90%. The degree of homology needed for thevariant to function in its intended capacity will depend upon theintended use of the sequence. It is well within the skill of a persontrained in this art to make mutational, insertional, and deletionalmutations which are designed to improve the function of the sequence orotherwise provide a methodological advantage.

PCR technology. Polymerase Chain Reaction (PCR) is a repetitive,enzymatic, primed synthesis of a nucleic acid sequence. This procedureis well known and commonly used by those skilled in this art (seeMullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki,Randall K., Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn,Henry A. Erlich, Norman Arnheim [1985] “Enzymatic Amplification ofβ-Globin Genomic Sequences and Restriction Site Analysis for Diagnosisof Sickle Cell Anemia,” Science 230:1350-1354.). PCR is based on theenzymatic amplification of a DNA fragment of interest that is flanked bytwo oligonucleotide primers that hybridize to opposite strands of thetarget sequence. The primers are oriented with the 3′ ends pointingtowards each other. Repeated cycles of heat denaturation of thetemplate, annealing of the primers to their complementary sequences, andextension of the annealed primers with a DNA polymerase result in theamplification of the segment defined by the 5′ ends of the PCR primers.Since the extension product of each primer can serve as a template forthe other primer, each cycle essentially doubles the amount of DNAfragment produced in the previous cycle. This results in the exponentialaccumulation of the specific target fragment, up to several million-foldin a few hours. By using a thermostable DNA polymerase such as Taqpolymerase, which is isolated from the thermophilic bacterium Thermusaquaticus, the amplification process can be completely automated. Otherenzymes which can be used are known to those skilled in the art.

The DNA sequences of the subject invention can be used as primers forPCR amplification. In performing PCR amplification, a certain degree ofmismatch can be tolerated between primer and template. Therefore,mutations, deletions, and insertions (especially additions ofnucleotides to the 5′ end) of the exemplified primers fall within thescope of the subject invention. Mutations, insertions and deletions canbe produced in a given primer by methods known to an ordinarily skilledartisan.

All of the references cited herein are hereby incorporated by reference.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Culturing of Bacillus Isolates Useful According to theInvention

The cellular host containing the Bacillus insecticidal gene may be grownin any convenient nutrient medium. These cells may then be harvested inaccordance with conventional ways. Alternatively, the cells can betreated prior to harvesting.

The Bacillus cells of the invention can be cultured using standard artmedia and fermentation techniques. During the fermentation cycle, thebacteria can be harvested by first separating the Bacillus vegetativecells, spores, crystals, and lysed cellular debris from the fermentationbroth by means well known in the art. Any Bacillus spores or crystalδ-endotoxins formed can be recovered employing well-known techniques andused as a conventional δ-endotoxin B.t. preparation. The supernatantfrom the fermentation process contains toxins of the present invention.The toxins are isolated and purified employing well-known techniques.

A subculture of Bacillus isolates, or mutants thereof, can be used toinoculate the following medium, known as TB broth:

Tryptone 12 g/l Yeast Extract 24 g/l Glycerol 4 g/l KH₂PO₄ 2.1 g/lK₂HPO₄ 14.7 g/l pH 7.4

The potassium phosphate was added to the autoclaved broth after cooling.Flasks were incubated at 30° C. on a rotary shaker at 250 rpm for 24-36hours.

The above procedure can be readily scaled up to large fermentors byprocedures well known in the art.

The Bacillus obtained in the above fermentation, can be isolated byprocedures well known in the art. A frequently-used procedure is tosubject the harvested fermentation broth to separation techniques, e.g.,centrifugation. In a specific embodiment, Bacillus proteins usefulaccording the present invention can be obtained from the supernatant.The culture supernatant containing the active protein(s) can be used inbioassays.

Alternatively, a subculture of Bacillus isolates, or mutants thereof,can be used to inoculate the following peptone, glucose, salts medium:

Bacto Peptone 7.5 g/l Glucose 1.0 g/l KH₂PO₄ 3.4 g/l K₂HPO₄ 4.35 g/lSalt Solution 5.0 ml/l CaCl₂ Solution 5.0 ml/l pH 7.2 Salts Solution(100 ml) MgSO₄.7H₂O 2.46 g MnSO₄.H₂O 0.04 g ZnSO₄.7H₂O 0.28 g FeSO₄.7H₂O0.40 g CaCl₂ Solution (100 ml) CaCl₂.2H₂O 3.66 g

The salts solution and CaCl₂ solution are filter-sterilized and added tothe autoclaved and cooked broth at the time of inoculation. Flasks areincubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors byprocedures well known in the art.

The Bacillus spores and/or crystals, obtained in the above fermentation,can be isolated by procedures well known in the art. A frequently-usedprocedure is to subject the harvested fermentation broth to separationtechniques, e.g., centrifugation.

EXAMPLE 2 Isolation and Preparation of Cellular DNA for PCR

DNA can be prepared from cells grown on Spizizen's agar, or otherminimal or enriched agar known to those skilled in the art, forapproximately 16 hours. Spizizen's casamino acid agar comprises 23.2 g/lSpizizen's minimal salts [(NH₄)₂SO₄, 120 g; K₂HPO₄, 840 g; KH₂PO₄, 360g; sodium citrate, 60 g; MgSO₄. 7H₂O, 12 g. Total: 1392 g]; 1.0 g/lvitamin-free casamino acids; 15.0 g/l Difco agar. In preparing the agar,the mixture was autoclaved for 30 minutes, then a sterile, 50% glucosesolution can be added to a final concentration of 0.5% (1/100 vol). Oncethe cells are grown for about 16 hours, an approximately 1 cm² patch ofcells can be scraped from the agar into 300 μl of 10 mM Tris-HCl (pH8.0)−1 mM EDTA. Proteinase K was added to 50 μg/ml and incubated at 55°C. for 15 minutes. Other suitable proteases lacking nuclease activitycan be used. The samples were then placed in a boiling water bath for 15minutes to inactivate the proteinase and denature the DNA. This alsoprecipitates unwanted components. The samples are then centrifuged at14,000×g in an Eppendorf microfuge at room temperature for 5 minutes toremove cellular debris. The supernatants containing crude DNA weretransferred to fresh tubes and frozen at −20° C. until used in PCRreactions.

Alternatively, total cellular DNA may be prepared from plate-grown cellsusing the QIAamp Tissue Kit from Qiagen (Santa Clarita, Calif.)following instructions from the manufacturer.

EXAMPLE 3 Primers Useful for Characterizing and/or Identifying ToxinGenes

The following set of PCR primers can be used to identify and/orcharacterize genes of the subject invention, which encode pesticidaltoxins:

GGRTTAMTTGGRTAYTATTT (SEQ ID NO. 3)

ATATCKWAYATTKGCATTTA (SEQ ID NO. 4)

Redundant nucleotide codes used throughout the subject disclosure are inaccordance with the IUPAC convention and include:

R=A or G

M=A or C

Y=C or T

K=G or T

W=A or T

EXAMPLE 4 Identification and Sequencing of Genes Encoding Novel SolubleProtein Toxins From Bacillus Strains

PCR using primers SEQ ID NO. 3 and SEQ ID NO. 4 was performed on totalcellular genomic DNA isolated from a broad range of B.t. strains. Thosesamples yielding an approximately 1 kb band were selected forcharacterization by DNA sequencing. Amplified DNA fragments were firstcloned into the PCR DNA TA-cloning plasmid vector, pCR2.1, as describedby the supplier (Invitrogen, San Diego, Calif.). Plasmids were isolatedfrom recombinant clones and tested for the presence of an approximately1 kbp insert by PCR using the plasmid vector primers, T3 and T7.

The following strains yielded the expected band of approximately 1000bp, thus indicating the presence of a MIS-type toxin gene: PS66D3,PS177C8, PS177I8, PS33F1, PS157C1 (157C1-A), PS201Z, PS31F2, andPS185Y2.

Plasmids were then isolated for use as sequencing templates using QIAGEN(Santa Clarita, Calif.) miniprep kits as described by the supplier.Sequencing reactions were performed using the Dye Terminator CycleSequencing Ready Reaction Kit from PE Applied Biosystems. Sequencingreactions were run on a ABI PRISM 377 Automated Sequencer. Sequence datawas collected, edited, and assembled using the ABI PRISM 377 Collection,Factura, and AutoAssembler software from PE ABI. DNA sequences weredetermined for portions of novel toxin genes from the followingisolates: PS66D3, PS177C8, PS177I8, PS33F1, PS157C1 (157C1-A), PS201Z,PS31F2, and PS185Y2. These nucleotide sequences are shown in SEQ ID NOS.5, 7, 9, 38, 33, 35, 36, and 37, respectively. Polypeptide sequenceswere deduced for portions of the encoded, novel soluble toxins from thefollowing isolates: PS66D3, PS177C8, PS177I8, and PS157C1 (toxin157C1-A). These nucleotide sequences are shown in SEQ ID NOS. 6, 8, 10,and 34, respectively.

EXAMPLE 5 Restriction Fragment Length Polymorphism (RFLP) of Toxins FromBacillus thuringiensis Strains

Total cellular DNA was prepared from various Bacillus thuriengensis(B.t.) strains grown to an optical density of 0.5-0.8 at 600 nm visiblelight. DNA was extracted using the Qiagen Genomic-tip 500/G kit andGenomic DNA Buffer Set according to protocol for Gram positive bacteria(Qiagen Inc.; Valencia, Calif.).

Standard Southern hybridizations using ³²P-lableled probes were used toidentifiy and characterize novel toxin genes within the total genomicDNA preparations. Prepared total genomic DNA was digested with variousrestriction enzymes, electrophoresed on a 1% agarose gel, andimmobilized on a supported nylon membrane using standard methods(Maniatis et al.).

PCR-amplified DNA fragments 1.0-1.1 kb in length were gel purified foruse as probes. Approximately 25 ng of each DNA fragment was used as atemplate for priming nascent DNA synthesis using DNA polymerase I Klenowfragment (New England Biolabs), random hexanucleotide primers(Boehringer Mannheim) and ³²PdCTP.

Each ³²P-lableled fragment served as a specific probe to itscorresponding genomic DNA blot. Hybridizations of immobilized DNA withrandomly labeled ³²P probes were performed in standard aqueous bufferconsisting of 5×SSPE, 5×Denhardt's solution, 0.5% SDS, 0.1 mg/ml at 65°C. overnight. Blots were washed under moderate stringency in 0.2×SSC,0.1% SDS at 65° C. and exposed to film. RFLP data showing specifichybridization bands containing all or part of the novel gene of interestwas obtained for each strain.

TABLE 3 (Strain)/ Probe Seq Gene Name I.D. Number RFLP Data (approximateband sizes) (PS)66D3 24 BamHI: 4.5 kbp, HindIII: >23 kbp, KpnI: 23 kbp,PstI: 15 kbp, XbaI: >23 kbp (PS)177I8 33 BamHI: >23 kbp, EcoRI: 10 kbp,HindIII: 2 kbp, SalI: >23 kbp, XabI: 3.5 kbp

In separate experiments, alternative probes for MIS and WAR genes wereused to detect novel toxin genes on Southern blots of genomic DNA by ³²Pautoradiography or by non-radioactive methods using the DIG nucleic acidlabeling and detection system (Boehringer Mannheim; Indianapolis, Ind.).DNA fragments approximately 2.6 kbp (PS177C8 MIS toxin gene; SEQ ID NO.7) and 1.3 kbp (PS177C8 WAR toxin gene; SEQ ID NO. 11) in length werePCR amplified from plasmid pMYC2450 using primers homologous to the 5′and 3′ ends of each respective gene. pMYC2450 is a recombinant plasmidcontaining the PS177C8 MIS and WAR genes on an approximately 14 kbp ClaIfragment in pHTBlueII (an E. coli/B. thuringiensis shuttle vectorcomprised of pBluescript S/K [Stratagene, La Jolla, Calif.] and thereplication origin from a resident B.t. plasmed [D. Lereclus et al.1989; FEMS Microbiology Letters 60:211-218]). These DNA fragments wereused as probes for MIS RFLP classes A through N and WAR RFLP classes Athrough L. RFLP data in Table 4 for class O was generated using MISfragments approximately 1636 bp amplified with primers S1-633F(CACTCAAAAAATGAAAAGGGAAA; SEQ ID NO. 39) and S1-2269R(CCGGTTTTATTGATGCTAC; SEQ ID NO. 40). RFLP data in Table 5 for class Mwas generated using WAR fragments approximately 495 bp amplified withprimers S2-501F (AGAACAATTTTTAGATAGGG; SEQ ID NO. 41) and S2-995R(TCCCTAAAGCATCAGAAATA; SEQ ID NO 42).

Fragments were gel purified and approximately 25 ng of each DNA fragmentwas randomly labeled with ³²P for radioactive detection or approximately300 ng of each DNA fragment was randomly labeled with the DIG High Primekit for nonradioactive detection. Hybridization of immobilized DNA withrandomly labeled ³²P probes were performed in standard formamideconditions: 50% formamide, 5×SSPE, 5×Denhardt's solution, 2% SDS, 0.1mg/ml sonicated sperm DNA at 42° C. overnight. Blots were washed underlow stringency in 2×SSC, 0.1% SDS at 42° C. and exposed to film. RFLPdata showing DNA bands containing all or part of the novel gene ofinterest was obtained for each strain.

RFLP data using MIS probes as discussed above were as follows:

TABLE 4 RFLP RFLP Data (approximate band Class Strain Name(s) size inbase pairs) A 177C8, 74H3, 66D3 HindIII: 2,454; 1,645 XbaI: 14,820;9,612; 8,138; 5,642; 1,440 B 177I8 HindIII: 2,454 XbaI: 3,500 (veryfaint 7,000) C 66D3 HindIII: 2,454 (faint 20,000) XbaI: 3,500 (faint7,000) D 28M, 31F2, 71G5, HindIII: 11,738; 7,614 71G7, 71I1, 71N1, XbaI:10,622; 6,030 146F, 185Y2, 201JJ7, KB73, KB68B46-2, KB71A35-4,KB71A116-1 D₁ 70B2, 71C2 HindIII: 11,738; 8,698; 7,614 XbaI: 11,354;10,622; 6,030 E KB68B51-2, KB68B55- HindIII: 6,975; 2,527 2 XbaI:10,000; 6,144 F KB53A49-4 HindIII: 5,766 XbaI: 6,757 G 86D1 HindIII:4,920 XbaI: 11,961 H HD573B, 33F1, 67B3 HindIII: 6,558; 1,978 XbaI:7,815; 6,558 I 205C, 40C1 HindIII: 6,752 XbaI: 4,618 J 130A3, 143A2,157C1 HindIII: 9,639; 3,943, 1,954; 1,210 XbaI: 7,005; 6,165; 4,480;3,699 K 201Z HindIII: 9,639; 4,339 XbaI: 7,232; 6,365 L 71G4 HindIII:7,005 XbaI: 9.639 M KB42A33-8, KB71A72- HindIII: 3,721 1, KB71A133-11XbaI: 3,274 N KB71A134-2 HindIII: 7,523 XbaI: 10,360; 3,490 OKB69A125-3, HindIII: 6,360; 3,726; 1,874; 1,098 KB69A127-7, XbaI: 6,360;5,893; 5,058; 3,726 KB69A136-2, KB7120-4

RFLP data using WAR probes as discussed above were as follows:

TABLE 5 RFLP RFLP Data (approximate band Class Strain Name(s) size inbase pairs) A 177C8, 74H3 HindIII: 3,659, 2,454, 606 XbaI: 5,457, 4,469,1,440, 966 B 177I8, 66D3 data unavailable C 28M, 31F2, 71G5, 71G7, 71I1,HindIII: 7,614 71N1, 146F, 185Y2, 201JJ7, XbaI: 10,982, 6,235 KB73,KB68B46-2, KB71A35- 4, KB71A116-1 C1 70B2, 71C2 HindIII: 8,698, 7,614XbaI: 11,354, 6,235 D KB68B51-2, KB68B55-2 HindIII: 7,200 Xbal: 6,342(and 11,225 for 51- 2)(and 9,888 for 55-2) E KB53A49-4 HindIII: 5,766XbaI: 6,757 F HD573B, 33F1, 67B3 HindIII: 3,348, 2,037 (and 6,558 forHD573B only) XbaI: 6,953 (and 7,815, 6,185 for HD573B only) G 205C, 40C1HindIII: 3,158 XbaI: 6,558, 2,809 H 130A3, 143A2, 157C1 HindIII: 4,339,3,361, 1,954, 660, 349 XbaI: 9.043, 4,203, 3,583, 2,958, 581, 464 I 201ZHindIII: 4,480, 3,819, 703 XbaI: 9,336, 3,256, 495 J 71G4 HindIII: 7,005XbaI: 9,639 K KB42A33-8, KB71A72-1, no hybridization signal KB71A133-l1L KB71A134-2 HindIII: 7,523 XbaI: 10,360 M KB69A125-3, KB69A127-7,HindIII: 5,058; 3,726; KB69A136-2, 3,198; 2,745; KB71A20-4 257 XbaI:5,255; 4,341; 3,452; 1,490; 474

EXAMPLE 6 Characterization and/or Identification of WAR Toxins

In a further embodiment of the subject invention, pesticidal toxins canbe characterized and/or identified by their level of reactivity withantibodies to pesticidal toxins exemplified herein. In a specificembodiment, antibodies can be raised to WAR toxins such as the toxinobtainable from PS177C8a. Other WAR toxins can then be identified and/orcharacterized by their reactivity with the antibodies. In a preferredembodiment, the antibodies are polyclonal antibodies. In this example,toxins with the greatest similarity to the 177C8a-WAR toxin would havethe greatest reactivity with the polyclonal antibodies. WAR toxins withgreater diversity react with the 177C8a polyclonal antibodies, but to alesser extent. Toxins which immunoreact with polyclonal antibodiesraised to the 177C8a WAR toxin can be obtained from, for example, theisolates designated PS177C8a, PS177I8, PS66D3, KB68B55-2, PS185Y2,KB53A49-4, KB68B51-2, PS31F2, PS74H3, PS28M, PS71G6, PS71G7, PS71I1,PS71N1, PS201JJ7, KB73, KB68B46-2, KB71A35-4, KB71A116-1, PS70B2,PS71C2, PS86D1, HD573B, PS33F1, PS67B3, PS205C, PS40C1, PS130A3,PS143A2, PS157C1, PS201Z, PS71G4, KB42A33-8, KB71A72-1, KB71A133-11,KB71A134-2, KB69A125-3, KB69A127-7, KB69A136-2, and KB71A20-4. IsolatesPS31F2 and KB68B46-2 show very weak antibody reactivity, suggestingadvantageous diversity.

EXAMPLE 7 Molecular Cloning and DNA Sequence Analysis of SolubleInsecticidal Protein (MIS and WAR) Genes From Bacillus thuringiensisStrain PS205C

Total cellular DNA was prepared from Bacillus thuringensis strain PS205Cgrown to an optical density of 0.5-0.8 at 600 nm visible light in LuriaBertani (LB) broth. DNA was extracted using the Qiagen Genomic-tip 500/Gkit and Genomic DNA Buffer Set according to the protocol for Grampositive bacteria (Qiagen Inc.; Valencia, Calif.). A PS205C cosmidlibrary was constructed in the SuperCos vector (Stratragene) usinginserts of PS205C total cellular DNA partially digested with Nde II.XL1-Blue cells (Stratagene) were transfected with packaged cosmids toobtain clones resistant to carbenicillin and kanamycin. 576 cosmidcolonies were grown in 96-well blocks in 1 ml LB+carbenicillin (100μg/ml)+kanamycin (50 μg/ml) at 37° C. for 18 hours and replica platedonto nylon filters for screening by hybridization.

A PCR amplicon containing approximately 1000 bp of the PS205C MIS genewas amplified from PS205 genomic DNA using primers SEQ ID NO. 3 and SEQID NO. 4 as described in Example 4. The DNA fragment was gel purifiedusing QiaexII extraction (Qiagen). The probe was radiolabeled with³²P-dCTP using the Prime-It II kit (Stratgene) and used in aqueoushybridization solution (6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1mg/ml denatured DNA) with the colony lift filters at 65° C. for 16hours. The colony lift filters were briefly washed 1× in 2×SSC/0. 1% SDSat room temperature followed by two additional washes for 10 minutes in0.5×SSC/0.1% SDS. The filters were then exposed to X-ray film for 5.5hours. One cosmid clone that hybridized strongly to the probe wasselected for further analysis. This cosmid clone was confirmed tocontain the MIS gene by PCR amplification with primers SEQ ID NO. 3 andSEQ ID NO. 4. This cosmid clone was designated as pMYC3105; recombinantE. coli XL-1Blue MR cells containing pMYC3105 are designated MR992.

A subculture of MR992 was deposited in the permanent collection of thePatent Culture Collection (NRRL), Regional Research Center, 1815 NorthUniversity Street, Peoria, Ill. 61604 USA on May 4, 1999. The accessionnumber is NRRL B-30124. A truncated plasmid clone for PS205C was alsodeposited on May 4, 1999. The accession number is NRRL B-30122.

To sequence the PS205C MIS and WAR genes, random transposon insertionsinto pMYC3105 were generated using the GPS-1 Genome Priming System andprotocols (New England Biolabs). The GPS2 trasposition vector encodingchloramphenicol resistance was chosen for selection of cosmidscontaining insertions. pMYC3105 cosmids that acquired transposons wereidentified by transformation and selection of E. coli XL1-Blue MR onmedia containing ampicillin, kanamycin and chloramphenicol. Cosmidtemplates were prepared from individual colonies for use as sequencingtemplates using the Multiscreen 96-well plasmid prep (Millipore). TheMIS and WAR toxin genes encoded by pMYC3105 were sequenced with GPS2primers using the ABI377 automated sequencing system and associatedsoftware. The MIS and WAR genes were found to be located next to oneanother in an apparent transcriptional operon. The nucleotide anddeduced polypeptide sequences are designated as new SEQ ID NOS. 43-46.

EXAMPLE 8 Molecular Cloning and DNA Sequence Analysis of SolubleInsecticidal Protein (MIS and WAR) Genes From Bacillus ThuringensisStrain PS31F2

a. Preparation and Cloning of Genomic DNA

Total cellular DNA was prepared from the Bacillus thuringensis strainPS31F2 grown to an optical density of 0.5-0.8 at 600 nm visible light inLuria Bertani (LB) broth. DNA was extracted using the Qiagen Genomic-tip500/G kit or Genomic-Tip 20/G and Genomic DNA Buffer Set (Qiagen Inc.;Valencia, Calif.) according to the protocol for Gram positive bacteria.

Lambda libraries containing total genomic DNA from Bacillus thuringensisstrain PS31F2 were prepared from DNA partially digested with NdeII.Partial NdeII restriction digests were electrophoresed on a 0.7% agarosegel and the region of the gel containing DNA fragments within the sizerange of 9-20 kbp was excised from the gel. DNA was electroeluted fromthe gel fragment in 0.1×TAE buffer at approximately 30 V for one hourand purified using Elutip-d columns (Schleicher and Schuell; Keene,N.H.).

Purified, fractionated DNA was ligated into BamHI-digested Lambda-GEM-11arms (Promega Corp., Madison, Wis.). Ligated DNA was then packaged intolambda phage using Gigapack III Gold packaging extract (StratageneCorp., La Jolla, Calif.). E. coli strain KW251 was infected withrecombinant phage and plated onto LB plates in LB top agarose. Plaqueswere lifted onto nitrocellulose filters and prepared for hybridizationusing standard methods (Maniatis, et al.). DNA fragments approximately1.1 kb (PS177C8 MIS) or 700 bp (PS177C8 WAR) in length were PCRamplified from plasmid pMYC2450 and used as the probes. Fragments weregel purified and approximately 25 ng of each DNA fragment was randomlylabeled with ³²P-dCTP. Hybridization of immobilized DNA with randomly³²P-labeled PS177C8 probes was performed in standard formamideconditions: 50% formamide, 5×SSPE, 5×Denhardt's solution, 2% SDS, 0.1mg/ml at 42° C. overnight. Blots were washed under low stringency in2×SSC, 0.1% SDS at 42° C. and exposed to film. Hybridizing plaques wereisolated from the plates and suspended in SM buffer. Phage DNA wasprepared using LambdaSorb phage adsorbent (Promega, Madison, Wis.). PCRusing the oligonucleotide primers SEQ ID NO. 3 and SEQ ID NO. 4 wasperformed using phage DNA templates to verify the presence of the targetgene. The PCR reactions yielded the expected 1 kb band in both DNAsamples confirming that those phage clones contain the gene of interest.For subcloning, phage DNA was digested with various enzymes,fractionated on a 1% agarose gel and blotted for Southern analysis.Southern analysis was performed as decribed above. A HindIII fragmentapproximately 8 kb in size was identified that contained the PS31F2toxin genes. This fragment was gel purified and cloned into the HindIIIsite of pBluescriptII (SK+); this plasmid clone is designated pMYC2610.The recombinant E. coli XL10Gold [pMYC2610] strain was designated MR983.

A subculture of MR983 was deposited in the permanent collection of thePatent Culture Collection (NRRL), Regional Research Center, 1815 NorthUniversity Street, Peoria, Ill. 61604 USA on May 4, 1999. The accessionnumber is NRRL B-30123.

b. DNA Sequencing

The pMYC2610 HindIII fragment containing the PS31F2 toxin genes wasisolated by restriction digestion, fractionation on a 0.7% agarose geland purification from the gel matrix using the QiaexII kit (Qiagen Inc.;Valencia, Calif.). Gel purified insert DNA was then digested separatelywith restriction enzymes AluI, MseI, or RsaI and fractionated on a 1%agarose gel. DNA fragments between 0.5 and 1.5 kb were excised from thegel and purified using the QiaexII kit. Recovered fragments were ligatedinto EcoRV digested pBluescriptII and transformed into E. coli XL10 Goldcells. Plasmid DNA was prepared from randomly chosen transformants,digested with NotI and ApaI to verify insert size and used as sequencingtemplates with primers homologous to plasmid vector sequences. Primerwalking was used to complete the sequence. Sequencing reactions wereperformed using dRhodamine or BigDye Sequencing kit (ABI Prism/PerkinElmer Applied Biosystems) and run on ABI 373 or 377 automatedsequencers. Data was analyzed using Factura, Autoassembler (ABI Prism)and Gentics Computer Group (Madison, Wis.) programs. The MIS and WARgenes were found to be located next to one another in an apparenttranscriptional operon. The WAR gene is 5′ to the MIS gene, and the twogenes are separated by 4 nucleotide bases.

The nucleotide sequences and deduced peptide sequences for the novel MISand WAR genes from PS31F2 are reported as new SEQ ID NOS. 47-50.

c. Subcloning and Transformation of B. thuringiensis

The PS31F2 toxin genes were subcloned on the 8 kbp HinDIII fragment frompMYC2610 into the E. coli/B.t. shuttle vector, pHT370 (O. Arantes and D.Lereclus. 1991. Gene 108: 115-119), for expression from the nativeBacillus promoter. The resulting plasmid construct was designatedpMYC2615. pMYC2415 plasmid DNA was prepared from recombinant E.coliXL10Gold for transformation into the acrystallierous (Cry-) B.t. host,CryB (A. Aronson, Purdue University, West Lafayette, Ind.), byelectroporation. The recombinant CryB [pMYC2615] strain was designatedMR558.

EXAMPLE 9 Molecular Cloning and DNA Sequence Analysis of a Novel SUPToxin Gene From Bacillus Thuringiensis strain KB59A4-6

Total cellular DNA was prepared from the Bacillus thuringensis strainKB59A4-6 grown to an optical density of 0.5-0.8 at 600 nm visible lightin Luria Bertani (LB) broth. DNA was extracted using the QiagenGenomic-tip 500/G kit and Genomic DNA Buffer Set according to theprotocol for Gram positive bacteria (Qiagen Inc.; Valencia, Calif.). DNAwas digested with HinDIII and run on 0.7% agarose gels for Southern blotanalysis by standard methods (Maniatis et al.). A PCR ampliconcontaining the SUP-like gene (SEQ ID NO. 1) from Javelin-90 genomic DNAwas obtained by using the oligos “3A-atg(GCTCTAGAAGGAGGTAACTTATGAACAAGAATAATACTAAATTAAGC) (SEQ ID NO. 51) and“3A-taa” (GGGGTACCTTACTTAATAGAGACATCG) (SEQ ID NO. 52). This DNAfragment was gel purified and labeled with radioactive ³²P-dCTP usingPrime-It II Random Primer Labeling Kit (Stratagene) for use as a probe.Hybridization of Southern blot filters was carried out in a solution of6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA at 42°C. overnight in a shaking water bath. The filters were subsequentlywashed in 1×SSPE and 0.1% SDS once at 25° C. followed by two additionalwashes at 37° C. Hybridized filters were then exposed to X-ray film at−80° C. An approximately 1 kbp HinDIII fragment of KB59A4-6 genomic DNAwas identified that hybridized to the Javelin 90 SUP probe.

A lambda library of KB59A4-6 genomic DNA was constructed as follows. DNAwas partially digested with Sau3A and size-fractionated on agarose gels.The region of the gel containing fragments between 9.0 and 23 kbp wasexcised and DNA was isolated by electroelution in 0.1×TAE bufferfollowed by purification over Elutip-d columns (Schleicher and Schuell,Keene, N.H.). Size-fractionated DNA inserts were ligated intoBamHI-digested Lambda-Gem 11 (Promega) and recombinant phage werepackaged using GigapackIII XL Packing Extract (Stratagene). Phage wereplated on E. coli VCS257 cells for screening by hybridization. Plaqueswere transferred to nylon filters and dried under vacuum at 80° C.Hybridization was then performed with the Javelin 90 Sup gene probe asdescribed above. One plaque that gave a positive signal was selectedusing a Pasteur pipette to obtain a plug. The plug was soaked over-nightat room temperature in 1 mL SM buffer+10 uL CHCl₃. Large-scale phage DNApreparations (Maniatis et al.) were obtained from liquid lysates of E.coli KW251 infected with this phage.

The KB59A4-6 toxin gene was subcloned into the E. coli/B. thuringiensisshuttle vector, pHT370 (O. Arantes and D. Lereclus. 1991. Gene 108:115-119), on an approximately 5.5 kbp SacI/XbaI fragment identified bySouthern hybridization. This plasmid subclone was designated pMYC2473.Recombinant E. coli XL10-Gold cells (Stratagene) containing thisconstruct are designated MR993. The insecticidal toxin gene wassequenced by primer walking using pMYC2473 plasmid and PCR amplicons asDNA templates. Sequencing reactions were performed using the DyeTerminator Cycle Sequencing Ready Reaction Kit from PE AppliedBiosystems and run on a ABI PRISM 377 Automated Sequencer. Sequence datawas analyzed using the PE ABI PRISM 377 Collection, Factura, andAutoAssembler software. The DNA sequence and deduced peptide sequence ofthe KB59A4-6 toxin are reported as new SEQ ID NOS. 53 and 54,respectively.

A subculture of MR993 was deposited in the permanent collection of thePatent Culture Collection (NRRL), Regional Research Center, 1815 NorthUniversity Street, Peoria, Ill. 61604 USA on May 4, 1999. The accessionnumber is NRRL B-30125.

EXAMPLE 10 Bioassays for Activity Against Lepidopterans and Coleopterans

Biological activity of the toxins and isolates of the subject inventioncan be confirmed using standard bioassay procedures. One such assay isthe budworm-bollworm (Heliothis virescens [Fabricius] and Helicoverpazea [Boddie]) assay. Lepidoptera bioassays were conducted with eithersurface application to artificial insect diet or diet incorporation ofsamples. All Lepidopteran insects were tested from the neonate stage tothe second instar. All assays were conducted with either toasted soyflour artificial diet or black cutworm artificial diet (BioServ,Frenchtown, N.J.).

Diet incorporation can be conducted by mixing the samples withartificial diet at a rate of 6 mL suspension plus 54 mL diet. Aftervortexing, this mixture is poured into plastic trays withcompartmentalized 3-ml wells (Nutrend Container Corporation,Jacksonville, Fla.). A water blank containing no B.t. serves as thecontrol. First instar larvae (USDA-ARS, Stoneville, Miss.) are placedonto the diet mixture. Wells are then sealed with Mylar sheeting(ClearLam Packaging, Ill.) using a tacking iron, and several pinholesare made in each well to provide gas exchange. Larvae were held at 25°C. for 6 days in a 14:10 (light:dark) holding room. Mortality andstunting are recorded after six days.

Bioassay by the top load method utilizes the same sample and dietpreparations as listed above. The samples are applied to the surface ofthe insect diet. In a specific embodiment, surface area ranged from 0.3to approximately 0.8 cm² depending on the tray size, 96 well tissueculture plates were used in addition to the format listed above.Following application, samples are allowed to air dry before insectinfestation. A water blank containing no B.t. can serve as the control.Eggs are applied to each treated well and were then sealed with Mylarsheeting (ClearLam Packaging, Ill.) using a tacking iron, and pinholesare made in each well to provide gas exchange. Bioassays are held at 25°C. for 7 days in a 14:10 (light:dark) or 28° C. for 4 days in a 14:10(light:dark) holding room. Mortality and insect stunting are recorded atthe end of each bioassay.

Another assay useful according to the subject invention is the Westerncorn rootworm assay. Samples can be bioassayed against neonate westerncorn rootworm larvae (Diabrotica virgifera virgifera) via top-loading ofsample onto an agar-based artificial diet at a rate of 160 ml/cm².Artificial diet can be dispensed into 0.78 cm² wells in 48-well tissueculture or similar plates and allowed to harden. After the dietsolidifies, samples are dispensed by pipette onto the diet surface.Excess liquid is then evaporated from the surface prior to transferringapproximately three neonate larvae per well onto the diet surface bycamel's hair brush. To prevent insect escape while allowing gasexchange, wells are heat-sealed with 2-mil punched polyester film with27HT adhesive (Oliver Products Company, Grand Rapids, Mich.). Bioassaysare held in darkness at 25° C., and mortality scored after four days.

Analogous bioassays can be performed by those skilled in the art toassess activity against other pests, such as the black cutworm (Agrotisipsilon).

Results are shown in Table 6.

TABLE 6 Genetics and function of concentrated B.t. supernatants screenedfor lepidopteran and coleopteran activity Approx. ca. 80-100 339 bp PCRTotal Protein kDa protein H. virescens H. zen Diabrotica Strain fragment(μg/cm²) (μg/cm²) % mortality Stunting % mortality Stunting % mortalityPS157C1 (#1) — 24 2  43 yes 13 yes — PS157C1 (#2) — 93 8 — — − — 40PS157C1 (#3) — 35 3 − — — — 18 Javelin 1990 ++ 43.2 3.6 100 yes 96 yesNT water 0-8 — 0-4 - 12

EXAMPLE 11 Results of Western Corn Rootworm Bioassays and FurtherCharacterization of the Toxins

Concentrated liquid supernatant solutions, obtained according to thesubject invention, were tested for activity against Western cornrootworm (WCRW). Supernatants from the following isolates were found tocause mortality against WCRW: PS31F2, PS66D3, PS177I8, KB53A49-4,KB68B46-2, KB68B51-2, KB68B55-2, and PS177C8.

Supernatants from the following isolates were also found to causemortality against WCRW: PS205A3, PS185V2, PS234E1, PS71G4, PS248N10,PS191A21, KB63B19-13, KB63B19-7, KB68B62-7, KB68B63-2, KB69A125-1,KB69A125-3, KB69A125-5, KB69A127-7, KB69A132-1, KB69B2-1, KB70B5-3,KB71A125-15, and KB71A35-6; it was confirmed that this activity was heatlabile. Furthermore, it was determined that the supernatants of thefollowing isolates did not react (yielded negative test results) withthe WAR antibody (see Example 12), and did not react with the MIS (SEQID NO. 31) and WAR (SEQ ID NO. 51) probes: PS205A3, PS185V2, PS234E1,PS71G4, PS248N10, PS191A21, KB63B19-13, KB63B19-7, KB68B62-7, KB68B63-2,KB69A125-1, KB69A125-5, KB69A132-1, KB69B2-1, KB70B5-3, KB71A125-15, andKB71A35-6; the supernatants of isolates KB69A125-3 and KB69A127-7yielded positive test results.

EXAMPLE 12 Culturing of 31F2 Clones and Bioassay of 31F2 Toxins onWestern Corn Rootworm (wCRW)

E. coli MR983 and the negative control strain MR948 (E. coli XL1-Blue[pSupercos]; vector control) were grown in 250 ml bottom baffled flaskscontaining 50 ml of DIFCO Terrific Broth medium. Cultures were incubatedin New Brunswick shaker agitating at 250 RPM, 30° C. for ˜23 hours.After 23 hours of incubation samples were aseptically taken to examinethe cultures under the microscope to check for presence of contaminants.30 ml of culture were dispensed into a 50 ml centrifuge tube andcentrifuged in a Sorvall centrifuge at 15,000 rpm for 20 minutes. The 1×supernatant was saved and submitted for bioassay against wCRW. Thepellet was resuspended 5× with 10 mM TRIS buffer, and was sonicatedprior to submission for bioassay against wCRW.

B.t. strain MR558 and the negative control MR539 (B.t. cry B[pHT BlueII]; vector control) were grown in the same manner except for theomission of glycerol from the Terrific Broth medium. B.t. cell pelletswere resuspended in water rather than buffer prior to sonication.

Assays for the E. coli clone MR983 and B. thuringiensis clone MR558containing the 31F2 toxin genes were conducted using the sameexperimental design as in Example 10 for western corn rootworm with thefollowing exceptions: Supernatant samples were top-loaded onto diet at adose of ˜160 ul/cm² . B.t. cellular pellet samples at a 5× concentrationwere top-loaded onto the diet at a dose of˜150 ul/ cm² for both clones,and at ˜75, and at doses of ˜35 ul/ cm2 for the MR558 B. thuringiensisclone (quantity of active toxin unknown for either clone). Approximately6-8 larvae were transferred onto the diet immediately after the samplehad evaporated. The bioassay plate was sealed with mylar sheeting usinga tacking iron and pinholes were made above each well to provide gasexchange. Both the MR983 and MR558 clones demonstrated degrees ofbioactivity (greater mortality) against western corn rootworm ascompared to the toxin-negative clones MR948 and MR539.

Table 7 presents the results showing the bioactivity of cloned PS31F2toxins against western corn rootworm.

TABLE 7 Percent Mortality of wCRW Toxin Rate Supernatant Pellet 5XPellet 5X Pellet 5X Strain genes => 160 ul/cm² 150 ul/cm² 75 ul/cm² 35ul/cm² MR983 31F2 7% 19% — — (4/56) (5/27) MR948 none 4% 26% — — (1/24)(6/23) MR983 31F2 3%(5/147) — 20% — (49/245) MR948 none 27%(19/70) — 51%— (79/154) MR983 31F2 13%(32/243) — 33% — (85/259) MR948 none 9%(14/155)— 20% — (55/273) MR558 31F2 35%(41/118) 88% 9%(9/100) 13%(13/97) (43/49)MR539 none 10%(14/134) 14% 15% 17%(19/111) (3/21) (17/11) MR558 31F23%(1/29) 35%(17/ 29%(15/52) 13%(7/55) 48) MR539 none 19%(5/27) 20%(9/31%(18/ 18%(9/49) 46) 57) MR558 31F2 13%(9/69) 38%(19/ 18%(15/15%(10/65) 50) 85) MR539 none 29%(16/55) 24%(14/ 14%(13/ 28%(18/64) 58)91) MR558 31F2 7%(5/74) 14% 17%(14/83) 11%(6/57) (9/66) MR539 none11%(9/79) 32% 9%(7/78) 15%(10/67) (19/59)

EXAMPLE 13 Target Pests

Toxins of the subject invention can be used, alone or in combinationwith other toxins, to control one or more non-mammalian pests. Thesepests may be, for example, those listed in Table 8. Activity can readilybe confirmed using the bioassays provided herein, adaptations of thesebioassays, and/or other bioassays well known to those skilled in theart.

TABLE 8 Target pest species ORDER/Common Name Latin Name LEPIDOPTERAEuropean Corn Borer Ostrinia nubilalis European Corn Borer resistant toOstrinia nubilalis Cry1A-class of toxins Black Cutworm Agrotis ipsilonFall Armyworm Spodoptera frugiperda Southwestern Corn Borer Diatraeagrandioseila Corn Earworm/Bollworm Helicoverpa zea Tobacco BudwormHeliothis virescens Tobacco Budworm resistant to Heliothis virescensCry1A-class of toxins Sunflower Head Moth Homeosoma ellectellum BandedSunflower Moth Cochylis hospes Argentine Looper Rachiplusia nu SpilosomaSpilosoma virginica Bertha Armyworm Mamestra configurata DiamondbackMoth Plutella xylostells Diamondback Moth resistant Plutella xylostellsto Cry1A-class of toxins COLEOPTERA Red Sunflower Seed Weevil Smicronyxfulvus Sunflower Stem Weevil Cylindrocopturus adspersus Sunflower BeetleZygoramma exclamationis Canola Flea Beetle Phyllotreta cruciferaeWestern Corn Rootworm Diabrotica virgifera virgifera DIPTERA Hessian FlyMayetiola destructor HOMOPTERA Greenbug Schizaphis graminum HEMIPTERALygus Bug Lygus lineolaris NEMATODA Heterodera glycines

EXAMPLE 14 Insertion of Toxin Genes Into Plants

One aspect of the subject invention is the transformation of plants withgenes encoding the insecticidal toxin of the present invention. Thetransformed plants are resistant to attack by the target pest.

Genes encoding pesticidal toxins, as disclosed herein, can be insertedinto plant cells using a variety of techniques which are well known inthe art. For example, a large number of cloning vectors comprising areplication system in E. coli and a marker that permits selection of thetransformed cells are available for preparation for the insertion offoreign genes into higher plants. The vectors comprise, for example,pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, thesequence encoding the Bacillus toxin can be inserted into the vector ata suitable restriction site. The resulting plasmid is used fortransformation into E. coli. The E. coli cells are cultivated in asuitable nutrient medium, then harvested and lysed. The plasmid isrecovered. Sequence analysis, restriction analysis, electrophoresis, andother biochemical-molecular biological methods are generally carried outas methods of analysis. After each manipulation, the DNA sequence usedcan be cleaved and joined to the next DNA sequence. Each plasmidsequence can be cloned in the same or other plasmids. Depending on themethod of inserting desired genes into the plant, other DNA sequencesmay be necessary. If, for example, the Ti or Ri plasmid is used for thetransformation of the plant cell, then at least the right border, butoften the right and the left border of the Ti or Ri plasmid T-DNA, hasto be joined as the flanking region of the genes to be inserted.

The use of T-DNA for the transformation of plant cells has beenintensively researched and sufficiently described in EP 120 516; Hoekema(1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters B.V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci.4:1-46; and An et al. (1985) EMBO J 4:277-287.

Once the inserted DNA has been integrated in the genome, it isrelatively stable there and, as a rule, does not come out again. Itnormally contains a selection marker that confers on the transformedplant cells resistance to a biocide or an antibiotic, such as kanamycin,G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. Theindividually employed marker should accordingly permit the selection oftransformed cells rather than cells that do not contain the insertedDNA.

A large number of techniques are available for inserting DNA into aplant host cell. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens or Agrobacterium rhizogenes astransformation agent, fusion, injection, biolistics (microparticlebombardment), or electroporation as well as other possible methods. IfAgrobacteria are used for the transformation, the DNA to be inserted hasto be cloned into special plasmids, namely either into an intermediatevector or into a binary vector. The intermediate vectors can beintegrated into the Ti or Ri plasmid by homologous recombination owingto sequences that are homologous to sequences in the T-DNA. The Ti or Riplasmid also comprises the vir region necessary for the transfer of theT-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria.The intermediate vector can be transferred into Agrobacteriumtumefaciens by means of a helper plasmid (conjugation). Binary vectorscan replicate themselves both in E. coli and in Agrobacteria. Theycomprise a selection marker gene and a linker or polylinker which areframed by the right and left T-DNA border regions. They can betransformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen.Genet. 163:181-187). The Agrobacterium used as host cell is to comprisea plasmid carrying a vir region. The vir region is necessary for thetransfer of the T-DNA into the plant cell. Additional T-DNA may becontained. The bacterium so transformed is used for the transformationof plant cells. Plant explants can advantageously be cultivated withAgrobacterium tumefaciens or Agrobacterium rhizogenes for the transferof the DNA into the plant cell. Whole plants can then be regeneratedfrom the infected plant material (for example, pieces of leaf, segmentsof stalk, roots, but also protoplasts or suspension-cultivated cells) ina suitable medium, which may contain antibiotics or biocides forselection. The plants so obtained can then be tested for the presence ofthe inserted DNA. No special demands are made of the plasmids in thecase of injection and electroporation. It is possible to use ordinaryplasmids, such as, for example, pUC derivatives. In biolistictransformation, plasmid DNA or linear DNA can be employed.

The transformed cells are regenerated into morphologically normal plantsin the usual manner. If a transformation event involves a germ linecell, then the inserted DNA and corresponding phenotypic trait(s) willbe transmitted to progeny plants. Such plants can be grown in the normalmanner and crossed with plants that have the same transformed hereditaryfactors or other hereditary factors. The resulting hybrid individualshave the corresponding phenotypic properties.

In a preferred embodiment of the subject invention, plants will betransformed with genes wherein the codon usage has been optimized forplants. See, for example, U.S. Pat. No. 5,380,831. Also, advantageously,plants encoding a truncated toxin will be used. The truncated toxintypically will encode about 55% to about 80% of the full length toxin.Methods for creating synthetic Bacillus genes for use in plants areknown in the art.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and of the appended claims.

54 2375 base pairs nucleic acid single linear DNA (genomic) Jav90 1ATGAACAAGA ATAATACTAA ATTAAGCACA AGAGCCTTAC CAAGTTTTAT TGATTATTTT 60AATGGCATTT ATGGATTTGC CACTGGTATC AAAGACATTA TGAACATGAT TTTTAAAACG 120GATACAGGTG GTGATCTAAC CCTAGACGAA ATTTTAAAGA ATCAGCAGTT ACTAAATGAT 180ATTTCTGGTA AATTGGATGG GGTGAATGGA AGCTTAAATG ATCTTATCGC ACAGGGAAAC 240TTAAATACAG AATTATCTAA GGAAATATTA AAAATTGCAA ATGAACAAAA TCAAGTTTTA 300AATGATGTTA ATAACAAACT CGATGCGATA AATACGATGC TTCGGGTATA TCTACCTAAA 360ATTACCTCTA TGTTGAGTGA TGTAATGAAA CAAAATTATG CGCTAAGTCT GCAAATAGAA 420TACTTAAGTA AACAATTGCA AGAGATTTCT GATAAGTTGG ATATTATTAA TGTAAATGTA 480CTTATTAACT CTACACTTAC TGAAATTACA CCTGCGTATC AAAGGATTAA ATATGTGAAC 540GAAAAATTTG AGGAATTAAC TTTTGCTACA GAAACTAGTT CAAAAGTAAA AAAGGATGGC 600TCTCCTGCAG ATATTCTTGA TGAGTTAACT GAGTTAACTG AACTAGCGAA AAGTGTAACA 660AAAAATGATG TGGATGGTTT TGAATTTTAC CTTAATACAT TCCACGATGT AATGGTAGGA 720AATAATTTAT TCGGGCGTTC AGCTTTAAAA ACTGCATCGG AATTAATTAC TAAAGAAAAT 780GTGAAAACAA GTGGCAGTGA GGTCGGAAAT GTTTATAACT TCTTAATTGT ATTAACAGCT 840CTGCAAGCAA AAGCTTTTCT TACTTTAACA ACATGCCGAA AATTATTAGG CTTAGCAGAT 900ATTGATTATA CTTCTATTAT GAATGAACAT TTAAATAAGG AAAAAGAGGA ATTTAGAGTA 960AACATCCTCC CTACACTTTC TAATACTTTT TCTAATCCTA ATTATGCAAA AGTTAAAGGA 1020AGTGATGAAG ATGCAAAGAT GATTGTGGAA GCTAAACCAG GACATGCATT GATTGGGTTT 1080GAAATTAGTA ATGATTCAAT TACAGTATTA AAAGTATATG AGGCTAAGCT AAAACAAAAT 1140TATCAAGTCG ATAAGGATTC CTTATCGGAA GTTATTTATG GTGATATGGA TAAATTATTG 1200TGCCCAGATC AATCTGAACA AATCTATTAT ACAAATAACA TAGTATTTCC AAATGAATAT 1260GTAATTACTA AAATTGATTT CACTAAAAAA ATGAAAACTT TAAGATATGA GGTAACAGCG 1320AATTTTTATG ATTCTTCTAC AGGAGAAATT GACTTAAATA AGAAAAAAGT AGAATCAAGT 1380GAAGCGGAGT ATAGAACGTT AAGTGCTAAT GATGATGGGG TGTATATGCC GTTAGGTGTC 1440ATCAGTGAAA CATTTTTGAC TCCGATTAAT GGGTTTGGCC TCCAAGCTGA TGAAAATTCA 1500AGATTAATTA CTTTAACATG TAAATCATAT TTAAGAGAAC TACTGCTAGC AACAGACTTA 1560AGCAATAAAG AAACTAAATT GATYGTCCCG CCAAGTGGTT TTATTAGCAA TATTGTAGAG 1620AACGGGTCCA TAGAAGAGGA CAATTTAGAG CCGTGGAAAG CAAATAATAA GAATGCGTAT 1680GTAGATCATA CAGGCGGAGT GAATGGAACT AAAGCTTTAT ATGTTCATAA GGACGGAGGA 1740ATTTCACAAT TTATTGGAGA TAAGTTAAAA CCGAAAACTG AGTATGTAAT CCAATATACT 1800GTTAAAGGAA AACCTTCTAT TCATTTAAAA GATGAAAATA CTGGATATAT TCATTATGAA 1860GATACAAATA ATAATTTAGA AGATTATCAA ACTATTAATA AACGTTTTAC TACAGGAACT 1920GATTTAAAGG GAGTGTATTT AATTTTAAAA AGTCAAAATG GAGATGAAGC TTGGGGAGAT 1980AACTTTATTA TTTTGGAAAT TAGTCCTTCT GAAAAGTTAT TAAGTCCAGA ATTAATTAAT 2040ACAAATAATT GGACGAGTAC GGGATCAACT AATATTAGCG GTAATACACT CACTCTTTAT 2100CAGGGAGGAC GAGGGATTCT AAAACAAAAC CTTCAATTAG ATAGTTTTTC AACTTATAGA 2160GTGTATTTTT CTGTGTCCGG AGATGCTAAT GTAAGGATTA GAAATTCTAG GGAAGTGTTA 2220TTTGAAAAAA GATATATGAG CGGTGCTAAA GATGTTTCTG AAATGTTCAC TACAAAATTT 2280GAGAAAGATA ACTTTTATAT AGAGCTTTCT CAAGGGAATA ATTTATATGG TGGTCCTATT 2340GTACATTTTT ACGATGTCTC TATTAAGTAA CCCAA 2375 790 amino acids amino acidsingle linear protein Jav90 2 Met Asn Lys Asn Asn Thr Lys Leu Ser ThrArg Ala Leu Pro Ser Phe 1 5 10 15 Ile Asp Tyr Phe Asn Gly Ile Tyr GlyPhe Ala Thr Gly Ile Lys Asp 20 25 30 Ile Met Asn Met Ile Phe Lys Thr AspThr Gly Gly Asp Leu Thr Leu 35 40 45 Asp Glu Ile Leu Lys Asn Gln Gln LeuLeu Asn Asp Ile Ser Gly Lys 50 55 60 Leu Asp Gly Val Asn Gly Ser Leu AsnAsp Leu Ile Ala Gln Gly Asn 65 70 75 80 Leu Asn Thr Glu Leu Ser Lys GluIle Leu Lys Ile Ala Asn Glu Gln 85 90 95 Asn Gln Val Leu Asn Asp Val AsnAsn Lys Leu Asp Ala Ile Asn Thr 100 105 110 Met Leu Arg Val Tyr Leu ProLys Ile Thr Ser Met Leu Ser Asp Val 115 120 125 Met Lys Gln Asn Tyr AlaLeu Ser Leu Gln Ile Glu Tyr Leu Ser Lys 130 135 140 Gln Leu Gln Glu IleSer Asp Lys Leu Asp Ile Ile Asn Val Asn Val 145 150 155 160 Leu Ile AsnSer Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile 165 170 175 Lys TyrVal Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr 180 185 190 SerSer Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Leu Asp Glu 195 200 205Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val 210 215220 Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly 225230 235 240 Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu LeuIle 245 250 255 Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly AsnVal Tyr 260 265 270 Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Lys AlaPhe Leu Thr 275 280 285 Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala AspIle Asp Tyr Thr 290 295 300 Ser Ile Met Asn Glu His Leu Asn Lys Glu LysGlu Glu Phe Arg Val 305 310 315 320 Asn Ile Leu Pro Thr Leu Ser Asn ThrPhe Ser Asn Pro Asn Tyr Ala 325 330 335 Lys Val Lys Gly Ser Asp Glu AspAla Lys Met Ile Val Glu Ala Lys 340 345 350 Pro Gly His Ala Leu Ile GlyPhe Glu Ile Ser Asn Asp Ser Ile Thr 355 360 365 Val Leu Lys Val Tyr GluAla Lys Leu Lys Gln Asn Tyr Gln Val Asp 370 375 380 Lys Asp Ser Leu SerGlu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu 385 390 395 400 Cys Pro AspGln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe 405 410 415 Pro AsnGlu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys 420 425 430 ThrLeu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly 435 440 445Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr 450 455460 Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val 465470 475 480 Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu GlnAla 485 490 495 Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser TyrLeu Arg 500 505 510 Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu ThrLys Leu Ile 515 520 525 Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val GluAsn Gly Ser Ile 530 535 540 Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala AsnAsn Lys Asn Ala Tyr 545 550 555 560 Val Asp His Thr Gly Gly Val Asn GlyThr Lys Ala Leu Tyr Val His 565 570 575 Lys Asp Gly Gly Ile Ser Gln PheIle Gly Asp Lys Leu Lys Pro Lys 580 585 590 Thr Glu Tyr Val Ile Gln TyrThr Val Lys Gly Lys Pro Ser Ile His 595 600 605 Leu Lys Asp Glu Asn ThrGly Tyr Ile His Tyr Glu Asp Thr Asn Asn 610 615 620 Asn Leu Glu Asp TyrGln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr 625 630 635 640 Asp Leu LysGly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Glu 645 650 655 Ala TrpGly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys 660 665 670 LeuLeu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly 675 680 685Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg 690 695700 Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg 705710 715 720 Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg AsnSer 725 730 735 Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala LysAsp Val 740 745 750 Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn PheTyr Ile Glu 755 760 765 Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pro IleVal His Phe Tyr 770 775 780 Asp Val Ser Ile Lys Pro 785 790 20 basepairs nucleic acid single linear DNA (genomic) 3 GGRTTAMTTG GRTAYTATTT20 20 base pairs nucleic acid single linear DNA (genomic) 4 ATATCKWAYATTKGCATTTA 20 1042 base pairs nucleic acid single linear DNA (genomic)66D3 5 TTAATTGGGT ACTATTTTAA AGGAAAAGAT TTTAATAATC TTACTATATT TGCTCCAACA60 CGTGAGAATA CTCTTATTTA TGATTTAGAA ACAGCGAATT CTTTATTAGA TAAGCAACAA 120CAAACCTATC AATCTATTCG TTGGATCGGT TTAATAAAAA GCAAAAAAGC TGGAGATTTT 180ACCTTTCAAT TATCGGATGA TGAGCATGCT ATTATAGAAA TCGATGGGAA AGTTATTTCG 240CAAAAAGGCC AAAAGAAACA AGTTGTTCAT TTAGAAAAAG ATAAATTAGT TCCCATCAAA 300ATTGAATATC AATCTGATAA AGCGTTAAAC CCAGATAGTC AAATGTTTAA AGAATTGAAA 360TTATTTAAAA TAAATAGTCA AAAACAATCT CAGCAAGTGC AACAAGACGA ATTGAGAAAT 420CCTGAATTTG GTAAAGAAAA AACTCAAACA TATTTAAAGA AAGCATCGAA AAGCAGCCTG 480TTTAGCAATA AAAGTAAACG AGATATAGAT GAAGATATAG ATGAGGATAC AGATACAGAT 540GGAGATGCCA TTCCTGATGT ATGGGAAGAA AATGGGTATA CCATCAAAGG AAGAGTAGCT 600GTTAAATGGG ACGAAGGATT AGCTGATAAG GGATATAAAA AGTTTGTTTC CAATCCTTTT 660AGACAGCACA CTGCTGGTGA CCCCTATAGT GACTATGAAA AGGCATCAAA AGATTTGGAT 720TTATCTAATG CAAAAGAAAC ATTTAATCCA TTGGTGGCTG CTTTTCCAAG TGTCAATGTT 780AGCTTGGAAA ATGTCACCAT ATCAAAAGAT GAAAATAAAA CTGCTGAAAT TGCGTCTACT 840TCATCGAATA ATTGGTCCTA TACAAATACA GAGGGGGCAT CTATTGAAGC TGGAATTGGA 900CCAGAAGGTT TGTTGTCTTT TGGAGTAAGT GCCAATTATC AACATTCTGA AACAGTGGCC 960AAAGAGTGGG GTACAACTAA GGGAGACGCA ACACAATATA ATACAGCTTC AGCAGGATAT 1020CTAAATGCCA ATGTACGATA TA 1042 347 amino acids amino acid single linearpeptide 66D3 6 Leu Ile Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asn LeuThr Ile 1 5 10 15 Phe Ala Pro Thr Arg Glu Asn Thr Leu Ile Tyr Asp LeuGlu Thr Ala 20 25 30 Asn Ser Leu Leu Asp Lys Gln Gln Gln Thr Tyr Gln SerIle Arg Trp 35 40 45 Ile Gly Leu Ile Lys Ser Lys Lys Ala Gly Asp Phe ThrPhe Gln Leu 50 55 60 Ser Asp Asp Glu His Ala Ile Ile Glu Ile Asp Gly LysVal Ile Ser 65 70 75 80 Gln Lys Gly Gln Lys Lys Gln Val Val His Leu GluLys Asp Lys Leu 85 90 95 Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Lys AlaLeu Asn Pro Asp 100 105 110 Ser Gln Met Phe Lys Glu Leu Lys Leu Phe LysIle Asn Ser Gln Lys 115 120 125 Gln Ser Gln Gln Val Gln Gln Asp Glu LeuArg Asn Pro Glu Phe Gly 130 135 140 Lys Glu Lys Thr Gln Thr Tyr Leu LysLys Ala Ser Lys Ser Ser Leu 145 150 155 160 Phe Ser Asn Lys Ser Lys ArgAsp Ile Asp Glu Asp Ile Asp Glu Asp 165 170 175 Thr Asp Thr Asp Gly AspAla Ile Pro Asp Val Trp Glu Glu Asn Gly 180 185 190 Tyr Thr Ile Lys GlyArg Val Ala Val Lys Trp Asp Glu Gly Leu Ala 195 200 205 Asp Lys Gly TyrLys Lys Phe Val Ser Asn Pro Phe Arg Gln His Thr 210 215 220 Ala Gly AspPro Tyr Ser Asp Tyr Glu Lys Ala Ser Lys Asp Leu Asp 225 230 235 240 LeuSer Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro 245 250 255Ser Val Asn Val Ser Leu Glu Asn Val Thr Ile Ser Lys Asp Glu Asn 260 265270 Lys Thr Ala Glu Ile Ala Ser Thr Ser Ser Asn Asn Trp Ser Tyr Thr 275280 285 Asn Thr Glu Gly Ala Ser Ile Glu Ala Gly Ile Gly Pro Glu Gly Leu290 295 300 Leu Ser Phe Gly Val Ser Ala Asn Tyr Gln His Ser Glu Thr ValAla 305 310 315 320 Lys Glu Trp Gly Thr Thr Lys Gly Asp Ala Thr Gln TyrAsn Thr Ala 325 330 335 Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr 340345 2645 base pairs nucleic acid single linear DNA (genomic) PS177C8a 7ATGAAGAAGA AGTTAGCAAG TGTTGTAACG TGTACGTTAT TAGCTCCTAT GTTTTTGAAT 60GGAAATGTGA ATGCTGTTTA CGCAGACAGC AAAACAAATC AAATTTCTAC AACACAGAAA 120AATCAACAGA AAGAGATGGA CCGAAAAGGA TTACTTGGGT ATTATTTCAA AGGAAAAGAT 180TTTAGTAATC TTACTATGTT TGCACCGACA CGTGATAGTA CTCTTATTTA TGATCAACAA 240ACAGCAAATA AACTATTAGA TAAAAAACAA CAAGAATATC AGTCTATTCG TTGGATTGGT 300TTGATTCAGA GTAAAGAAAC GGGAGATTTC ACATTTAACT TATCTGAGGA TGAACAGGCA 360ATTATAGAAA TCAATGGGAA AATTATTTCT AATAAAGGGA AAGAAAAGCA AGTTGTCCAT 420TTAGAAAAAG GAAAATTAGT TCCAATCAAA ATAGAGTATC AATCAGATAC AAAATTTAAT 480ATTGACAGTA AAACATTTAA AGAACTTAAA TTATTTAAAA TAGATAGTCA AAACCAACCC 540CAGCAAGTCC AGCAAGATGA ACTGAGAAAT CCTGAATTTA ACAAGAAAGA ATCACAGGAA 600TTCTTAGCGA AACCATCGAA AATAAATCTT TTCACTCAAA AAATGAAAAG GGAAATTGAT 660GAAGACACGG ATACGGATGG GGACTCTATT CCTGACCTTT GGGAAGAAAA TGGGTATACG 720ATTCAAAATA GAATCGCTGT AAAGTGGGAC GATTCTYTAG CAAGTAAAGG GTATACGAAA 780TTTGTTTCAA ATCCGCTAGA AAGTCACACA GTTGGTGATC CTTATACAGA TTATGAAAAG 840GCAGCAAGAG ACCTAGATTT GTCAAATGCA AAGGAAACGT TTAACCCATT GGTAGCTGCT 900TTTCCAAGTG TGAATGTTAG TATGGAAAAG GTGATATTAT CACCAAATGA AAATTTATCC 960AATAGTGTAG AGTCTCATTC ATCCACGAAT TGGTCTTATA CAAATACAGA AGGTGCTTCT 1020GTTGAAGCGG GGATTGGACC AAAAGGTATT TCGTTCGGAG TTAGCGTAAA CTATCAACAC 1080TCTGAAACAG TTGCACAAGA ATGGGGAACA TCTACAGGAA ATACTTCGCA ATTCAATACG 1140GCTTCAGCGG GATATTTAAA TGCAAATGTT CGATATAACA ATGTAGGAAC TGGTGCCATC 1200TACGATGTAA AACCTACAAC AAGTTTTGTA TTAAATAACG ATACTATCGC AACTATTACG 1260GCGAAATCTA ATTCTACAGC CTTAAATATA TCTCCTGGAG AAAGTTACCC GAAAAAAGGA 1320CAAAATGGAA TCGCAATAAC ATCAATGGAT GATTTTAATT CCCATCCGAT TACATTAAAT 1380AAAAAACAAG TAGATAATCT GCTAAATAAT AAACCTATGA TGTTGGAAAC AAACCAAACA 1440GATGGTGTTT ATAAGATAAA AGATACACAT GGAAATATAG TAACTGGCGG AGAATGGAAT 1500GGTGTCATAC AACAAATCAA GGCTAAAACA GCGTCTATTA TTGTGGATGA TGGGGAACGT 1560GTAGCAGAAA AACGTGTAGC GGCAAAAGAT TATGAAAATC CAGAAGATAA AACACCGTCT 1620TTAACTTTAA AAGATGCCCT GAAGCTTTCA TATCCAGATG AAATAAAAGA AATAGAGGGA 1680TTATTATATT ATAAAAACAA ACCGATATAC GAATCGAGCG TTATGACTTA CTTAGATGAA 1740AATACAGCAA AAGAAGTGAC CAAACAATTA AATGATACCA CTGGGAAATT TAAAGATGTA 1800AGTCATTTAT ATGATGTAAA ACTGACTCCA AAAATGAATG TTACAATCAA ATTGTCTATA 1860CTTTATGATA ATGCTGAGTC TAATGATAAC TCAATTGGTA AATGGACAAA CACAAATATT 1920GTTTCAGGTG GAAATAACGG AAAAAAACAA TATTCTTCTA ATAATCCGGA TGCTAATTTG 1980ACATTAAATA CAGATGCTCA AGAAAAATTA AATAAAAATC GTACTATTAT ATAAGTTTAT 2040ATATGAAGTC AGAAAAAAAC ACACAATGTG AGATTACTAT AGATGGGGAG ATTTATCCGA 2100TCACTACAAA AACAGTGAAT GTGAATAAAG ACAATTACAA AAGATTAGAT ATTATAGCTC 2160ATAATATAAA AAGTAATCCA ATTTCTTCAA TTCATATTAA AACGAATGAT GAAATAACTT 2220TATTTTGGGA TGATATTTCT ATAACAGATG TAGCATCAAT AAAACCGGAA AATTTAACAG 2280ATTCAGAAAT TAAACAGATT TATAGTAGGT ATGGTATTAA GTTAGAAGAT GGAATCCTTA 2340TTGATAAAAA AGGTGGGATT CATTATGGTG AATTTATTAA TGAAGCTAGT TTTAATATTG 2400AACCATTGCA AAATTATGTG ACAAAATATA AAGTTACTTA TAGTAGTGAG TTAGGACAAA 2460ACGTGAGTGA CACACTTGAA AGTGATAAAA TTTACAAGGA TGGGACAATT AAATTTGATT 2520TTACAAAATA TAGTRAAAAT GAACAAGGAT TATTTTATGA CAGTGGATTA AATTGGGACT 2580TTAAAATTAA TGCTATTACT TATGATGGTA AAGAGATGAA TGTTTTTCAT AGATATAATA 2640AATAG 2645 881 amino acids amino acid single linear peptide PS177C8a 8Met Lys Lys Lys Leu Ala Ser Val Val Thr Cys Thr Leu Leu Ala Pro 1 5 1015 Met Phe Leu Asn Gly Asn Val Asn Ala Val Tyr Ala Asp Ser Lys Thr 20 2530 Asn Gln Ile Ser Thr Thr Gln Lys Asn Gln Gln Lys Glu Met Asp Arg 35 4045 Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Ser Asn Leu 50 5560 Thr Met Phe Ala Pro Thr Arg Asp Ser Thr Leu Ile Tyr Asp Gln Gln 65 7075 80 Thr Ala Asn Lys Leu Leu Asp Lys Lys Gln Gln Glu Tyr Gln Ser Ile 8590 95 Arg Trp Ile Gly Leu Ile Gln Ser Lys Glu Thr Gly Asp Phe Thr Phe100 105 110 Asn Leu Ser Glu Asp Glu Gln Ala Ile Ile Glu Ile Asn Gly LysIle 115 120 125 Ile Ser Asn Lys Gly Lys Glu Lys Gln Val Val His Leu GluLys Gly 130 135 140 Lys Leu Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp ThrLys Phe Asn 145 150 155 160 Ile Asp Ser Lys Thr Phe Lys Glu Leu Lys LeuPhe Lys Ile Asp Ser 165 170 175 Gln Asn Gln Pro Gln Gln Val Gln Gln AspGlu Leu Arg Asn Pro Glu 180 185 190 Phe Asn Lys Lys Glu Ser Gln Glu PheLeu Ala Lys Pro Ser Lys Ile 195 200 205 Asn Leu Phe Thr Gln Lys Met LysArg Glu Ile Asp Glu Asp Thr Asp 210 215 220 Thr Asp Gly Asp Ser Ile ProAsp Leu Trp Glu Glu Asn Gly Tyr Thr 225 230 235 240 Ile Gln Asn Arg IleAla Val Lys Trp Asp Asp Ser Leu Ala Ser Lys 245 250 255 Gly Tyr Thr LysPhe Val Ser Asn Pro Leu Glu Ser His Thr Val Gly 260 265 270 Asp Pro TyrThr Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser 275 280 285 Asn AlaLys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro Ser Val 290 295 300 AsnVal Ser Met Glu Lys Val Ile Leu Ser Pro Asn Glu Asn Leu Ser 305 310 315320 Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr 325330 335 Glu Gly Ala Ser Val Glu Ala Gly Ile Gly Pro Lys Gly Ile Ser Phe340 345 350 Gly Val Ser Val Asn Tyr Gln His Ser Glu Thr Val Ala Gln GluTrp 355 360 365 Gly Thr Ser Thr Gly Asn Thr Ser Gln Phe Asn Thr Ala SerAla Gly 370 375 380 Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly ThrGly Ala Ile 385 390 395 400 Tyr Asp Val Lys Pro Thr Thr Ser Phe Val LeuAsn Asn Asp Thr Ile 405 410 415 Ala Thr Ile Thr Ala Lys Ser Asn Ser ThrAla Leu Asn Ile Ser Pro 420 425 430 Gly Glu Ser Tyr Pro Lys Lys Gly GlnAsn Gly Ile Ala Ile Thr Ser 435 440 445 Met Asp Asp Phe Asn Ser His ProIle Thr Leu Asn Lys Lys Gln Val 450 455 460 Asp Asn Leu Leu Asn Asn LysPro Met Met Leu Glu Thr Asn Gln Thr 465 470 475 480 Asp Gly Val Tyr LysIle Lys Asp Thr His Gly Asn Ile Val Thr Gly 485 490 495 Gly Glu Trp AsnGly Val Ile Gln Gln Ile Lys Ala Lys Thr Ala Ser 500 505 510 Ile Ile ValAsp Asp Gly Glu Arg Val Ala Glu Lys Arg Val Ala Ala 515 520 525 Lys AspTyr Glu Asn Pro Glu Asp Lys Thr Pro Ser Leu Thr Leu Lys 530 535 540 AspAla Leu Lys Leu Ser Tyr Pro Asp Glu Ile Lys Glu Ile Glu Gly 545 550 555560 Leu Leu Tyr Tyr Lys Asn Lys Pro Ile Tyr Glu Ser Ser Val Met Thr 565570 575 Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Thr Lys Gln Leu Asn Asp580 585 590 Thr Thr Gly Lys Phe Lys Asp Val Ser His Leu Tyr Asp Val LysLeu 595 600 605 Thr Pro Lys Met Asn Val Thr Ile Lys Leu Ser Ile Leu TyrAsp Asn 610 615 620 Ala Glu Ser Asn Asp Asn Ser Ile Gly Lys Trp Thr AsnThr Asn Ile 625 630 635 640 Val Ser Gly Gly Asn Asn Gly Lys Lys Gln TyrSer Ser Asn Asn Pro 645 650 655 Asp Ala Asn Leu Thr Leu Asn Thr Asp AlaGln Glu Lys Leu Asn Lys 660 665 670 Asn Arg Asp Tyr Tyr Ile Ser Leu TyrMet Lys Ser Glu Lys Asn Thr 675 680 685 Gln Cys Glu Ile Thr Ile Asp GlyGlu Ile Tyr Pro Ile Thr Thr Lys 690 695 700 Thr Val Asn Val Asn Lys AspAsn Tyr Lys Arg Leu Asp Ile Ile Ala 705 710 715 720 His Asn Ile Lys SerAsn Pro Ile Ser Ser Ile His Ile Lys Thr Asn 725 730 735 Asp Glu Ile ThrLeu Phe Trp Asp Asp Ile Ser Ile Thr Asp Val Ala 740 745 750 Ser Ile LysPro Glu Asn Leu Thr Asp Ser Glu Ile Lys Gln Ile Tyr 755 760 765 Ser ArgTyr Gly Ile Lys Leu Glu Asp Gly Ile Leu Ile Asp Lys Lys 770 775 780 GlyGly Ile His Tyr Gly Glu Phe Ile Asn Glu Ala Ser Phe Asn Ile 785 790 795800 Glu Pro Leu Gln Asn Tyr Val Thr Lys Tyr Lys Val Thr Tyr Ser Ser 805810 815 Glu Leu Gly Gln Asn Val Ser Asp Thr Leu Glu Ser Asp Lys Ile Tyr820 825 830 Lys Asp Gly Thr Ile Lys Phe Asp Phe Thr Lys Tyr Ser Xaa AsnGlu 835 840 845 Gln Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe LysIle Asn 850 855 860 Ala Ile Thr Tyr Asp Gly Lys Glu Met Asn Val Phe HisArg Tyr Asn 865 870 875 880 Lys 1022 base pairs nucleic acid singlelinear DNA (genomic) 177I8 9 TGGATTAATT GGGTATTATT TCAAAGGAAA AGATTTTAATAATCTTACTA TGTTTGCACC 60 GACACGTGAT AATACCCTTA TGTATGACCA ACAAACAGCGAATGCATTAT TAGATAAAAA 120 ACAACAAGAA TATCAGTCCA TTCGTTGGAT TGGTTTGATTCAGAGTAAAG AAACGGGCGA 180 TTTCACATTT AACTTATCAA AGGATGAACA GGCAATTATAGAAATCGATG GGAAAATCAT 240 TTCTAATAAA GGGAAAGAAA AGCAAGTTGT CCATTTAGAAAAAGAAAAAT TAGTTCCAAT 300 CAAAATAGAG TATCAATCAG ATACGAAATT TAATATTGATAGTAAAACAT TTAAAGAACT 360 TAAATTATTT AAAATAGATA GTCAAAACCA ATCTCAACAAGTTCAACTGA GAAACCCTGA 420 ATTTAACAAA AAAGAATCAC AGGAATTTTT AGCAAAAGCATCAAAAACAA ACCTTTTTAA 480 GCAAAAAATG AAAAGAGATA TTGATGAAGA TACGGATACAGATGGAGACT CCATTCCTGA 540 TCTTTGGGAA GAAAATGGGT ACACGATTCA AAATAAAGTTGCTGTCAAAT GGGATGATTC 600 GCTAGCAAGT AAGGGATATA CAAAATTTGT TTCGAATCCATTAGACAGCC ACACAGTTGG 660 CGATCCCTAT ACTGATTATG AAAAGGCCGC AAGGGATTTAGATTTATCAA ATGCAAAGGA 720 AACGTTCAAC CCATTGGTAG CTGCTTTYCC AAGTGTGAATGTTAGTATGG AAAAGGTGAT 780 ATTATCACCA AATGAAAATT TATCCAATAG TGTAGAGTCTCATTCATCCA CGAATTGGTC 840 TTATACGAAT ACAGAAGGAG CTTCCATTGA AGCTGGTGGCGGTCCATTAG GCCTTTCTTT 900 TGGAGTGAGT GTTAATTATC AACACTCTGA AACAGTTGCACAAGAATGGG GAACATCTAC 960 AGGAAATACT TCACAATTCA ATACGGCTTC AGCGGGATATTTAAATGCCA ATATACGATA 1020 TA 1022 340 amino acids amino acid singlelinear peptide 177I8 10 Gly Leu Ile Gly Tyr Tyr Phe Lys Gly Lys Asp PheAsn Asn Leu Thr 1 5 10 15 Met Phe Ala Pro Thr Arg Asp Asn Thr Leu MetTyr Asp Gln Gln Thr 20 25 30 Ala Asn Ala Leu Leu Asp Lys Lys Gln Gln GluTyr Gln Ser Ile Arg 35 40 45 Trp Ile Gly Leu Ile Gln Ser Lys Glu Thr GlyAsp Phe Thr Phe Asn 50 55 60 Leu Ser Lys Asp Glu Gln Ala Ile Ile Glu IleAsp Gly Lys Ile Ile 65 70 75 80 Ser Asn Lys Gly Lys Glu Lys Gln Val ValHis Leu Glu Lys Glu Lys 85 90 95 Leu Val Pro Ile Lys Ile Glu Tyr Gln SerAsp Thr Lys Phe Asn Ile 100 105 110 Asp Ser Lys Thr Phe Lys Glu Leu LysLeu Phe Lys Ile Asp Ser Gln 115 120 125 Asn Gln Ser Gln Gln Val Gln LeuArg Asn Pro Glu Phe Asn Lys Lys 130 135 140 Glu Ser Gln Glu Phe Leu AlaLys Ala Ser Lys Thr Asn Leu Phe Lys 145 150 155 160 Gln Lys Met Lys ArgAsp Ile Asp Glu Asp Thr Asp Thr Asp Gly Asp 165 170 175 Ser Ile Pro AspLeu Trp Glu Glu Asn Gly Tyr Thr Ile Gln Asn Lys 180 185 190 Val Ala ValLys Trp Asp Asp Ser Leu Ala Ser Lys Gly Tyr Thr Lys 195 200 205 Phe ValSer Asn Pro Leu Asp Ser His Thr Val Gly Asp Pro Tyr Thr 210 215 220 AspTyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser Asn Ala Lys Glu 225 230 235240 Thr Phe Asn Pro Leu Val Ala Ala Xaa Pro Ser Val Asn Val Ser Met 245250 255 Glu Lys Val Ile Leu Ser Pro Asn Glu Asn Leu Ser Asn Ser Val Glu260 265 270 Ser His Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr Glu Gly AlaSer 275 280 285 Ile Glu Ala Gly Gly Gly Pro Leu Gly Leu Ser Phe Gly ValSer Val 290 295 300 Asn Tyr Gln His Ser Glu Thr Val Ala Gln Glu Trp GlyThr Ser Thr 305 310 315 320 Gly Asn Thr Ser Gln Phe Asn Thr Ala Ser AlaGly Tyr Leu Asn Ala 325 330 335 Asn Ile Arg Tyr 340 1341 base pairsnucleic acid single linear DNA (genomic) PS177C8a 11 ATGTTTATGGTTTCTAAAAA ATTACAAGTA GTTACTAAAA CTGTATTGCT TAGTACAGTT 60 TTCTCTATATCTTTATTAAA TAATGAAGTG ATAAAAGCTG AACAATTAAA TATAAATTCT 120 CAAAGTAAATATACTAACTT GCAAAATCTA AAAATCACTG ACAAGGTAGA GGATTTTAAA 180 GAAGATAAGGAAAAAGCGAA AGAATGGGGG AAAGAAAAAG AAAAAGAGTG GAAACTAACT 240 GCTACTGAAAAAGGAAAAAT GAATAATTTT TTAGATAATA AAAATGATAT AAAGACAAAT 300 TATAAAGAAATTACTTTTTC TATGGCAGGC TCATTTGAAG ATGAAATAAA AGATTTAAAA 360 GAAATTGATAAGATGTTTGA TAAAACCAAT CTATCAAATT CTATTATCAC CTATAAAAAT 420 GTGGAACCGACAACAATTGG ATTTAATAAA TCTTTAACAG AAGGTAATAC GATTAATTCT 480 GATGCAATGGCACAGTTTAA AGAACAATTT TTAGATAGGG ATATTAAGTT TGATAGTTAT 540 CTAGATACGCATTTAACTGC TCAACAAGTT TCCAGTAAAG AAAGAGTTAT TTTGAAGGTT 600 ACGGTTCCGAGTGGGAAAGG TTCTACTACT CCAACAAAAG CAGGTGTCAT TTTAAATAAT 660 AGTGAATACAAAATGCTCAT TGATAATGGG TATATGGTCC ATGTAGATAA GGTATCAAAA 720 GTGGTGAAAAAAGGGGTGGA GTGCTTACAA ATTGAAGGGA CTTTAAAAAA GAGTCTTGAC 780 TTTAAAAATGATATAAATGC TGAAGCGCAT AGCTGGGGTA TGAAGAATTA TGAAGAGTGG 840 GCTAAAGATTTAACCGATTC GCAAAGGGAA GCTTTAGATG GGTATGCTAG GCAAGATTAT 900 AAAGAAATCAATAATTATTT AAGAAATCAA GGCGGAAGTG GAAATGAAAA ACTAGATGCT 960 CAAATAAAAAATATTTCTGA TGCTTTAGGG AAGAAACCAA TACCGGAAAA TATTACTGTG 1020 TATAGATGGTGTGGCATGCC GGAATTTGGT TATCAAATTA GTGATCCGTT ACCTTCTTTA 1080 AAAGATTTTGAAGAACAATT TTTAAATACA ATCAAAGAAG ACAAAGGATA TATGAGTACA 1140 AGCTTATCGAGTGAACGTCT TGCAGCTTTT GGATCTAGAA AAATTATATT ACGATTACAA 1200 GTTCCGAAAGGAAGTACGGG TGCGTATTTA AGTGCCATTG GTGGATTTGC AAGTGAAAAA 1260 GAGATCCTACTTGATAAAGA TAGTAAATAT CATATTGATA AAGTAACAGA GGTAATTATT 1320 AAGGTGTTAAGCGATATGTA G 1341 446 amino acids amino acid single linear peptidePS177C8a 12 Met Phe Met Val Ser Lys Lys Leu Gln Val Val Thr Lys Thr ValLeu 1 5 10 15 Leu Ser Thr Val Phe Ser Ile Ser Leu Leu Asn Asn Glu ValIle Lys 20 25 30 Ala Glu Gln Leu Asn Ile Asn Ser Gln Ser Lys Tyr Thr AsnLeu Gln 35 40 45 Asn Leu Lys Ile Thr Asp Lys Val Glu Asp Phe Lys Glu AspLys Glu 50 55 60 Lys Ala Lys Glu Trp Gly Lys Glu Lys Glu Lys Glu Trp LysLeu Thr 65 70 75 80 Ala Thr Glu Lys Gly Lys Met Asn Asn Phe Leu Asp AsnLys Asn Asp 85 90 95 Ile Lys Thr Asn Tyr Lys Glu Ile Thr Phe Ser Met AlaGly Ser Phe 100 105 110 Glu Asp Glu Ile Lys Asp Leu Lys Glu Ile Asp LysMet Phe Asp Lys 115 120 125 Thr Asn Leu Ser Asn Ser Ile Ile Thr Tyr LysAsn Val Glu Pro Thr 130 135 140 Thr Ile Gly Phe Asn Lys Ser Leu Thr GluGly Asn Thr Ile Asn Ser 145 150 155 160 Asp Ala Met Ala Gln Phe Lys GluGln Phe Leu Asp Arg Asp Ile Lys 165 170 175 Phe Asp Ser Tyr Leu Asp ThrHis Leu Thr Ala Gln Gln Val Ser Ser 180 185 190 Lys Glu Arg Val Ile LeuLys Val Thr Val Pro Ser Gly Lys Gly Ser 195 200 205 Thr Thr Pro Thr LysAla Gly Val Ile Leu Asn Asn Ser Glu Tyr Lys 210 215 220 Met Leu Ile AspAsn Gly Tyr Met Val His Val Asp Lys Val Ser Lys 225 230 235 240 Val ValLys Lys Gly Val Glu Cys Leu Gln Ile Glu Gly Thr Leu Lys 245 250 255 LysSer Leu Asp Phe Lys Asn Asp Ile Asn Ala Glu Ala His Ser Trp 260 265 270Gly Met Lys Asn Tyr Glu Glu Trp Ala Lys Asp Leu Thr Asp Ser Gln 275 280285 Arg Glu Ala Leu Asp Gly Tyr Ala Arg Gln Asp Tyr Lys Glu Ile Asn 290295 300 Asn Tyr Leu Arg Asn Gln Gly Gly Ser Gly Asn Glu Lys Leu Asp Ala305 310 315 320 Gln Ile Lys Asn Ile Ser Asp Ala Leu Gly Lys Lys Pro IlePro Glu 325 330 335 Asn Ile Thr Val Tyr Arg Trp Cys Gly Met Pro Glu PheGly Tyr Gln 340 345 350 Ile Ser Asp Pro Leu Pro Ser Leu Lys Asp Phe GluGlu Gln Phe Leu 355 360 365 Asn Thr Ile Lys Glu Asp Lys Gly Tyr Met SerThr Ser Leu Ser Ser 370 375 380 Glu Arg Leu Ala Ala Phe Gly Ser Arg LysIle Ile Leu Arg Leu Gln 385 390 395 400 Val Pro Lys Gly Ser Thr Gly AlaTyr Leu Ser Ala Ile Gly Gly Phe 405 410 415 Ala Ser Glu Lys Glu Ile LeuLeu Asp Lys Asp Ser Lys Tyr His Ile 420 425 430 Asp Lys Val Thr Glu ValIle Ile Lys Val Leu Ser Asp Met 435 440 445 21 base pairs nucleic acidsingle linear DNA (genomic) 13 GCTGATGAAC CATTTAATGC C 21 22 base pairsnucleic acid single linear DNA (genomic) 14 CTCTTTAAAG TAGATACTAA GC 2224 base pairs nucleic acid single linear DNA (genomic) 15 GATGAGAACTTATCAAATAG TATC 24 33 base pairs nucleic acid single linear DNA(genomic) 16 CGAATTCTTT ATTAGATAAG CAACAACAAA CCT 33 24 base pairsnucleic acid single linear DNA (genomic) 17 GTTATTTCGC AAAAAGGCCA AAAG24 31 base pairs nucleic acid single linear DNA (genomic) 18 GAATATCAATCTGATAAAGC GTTAAACCCA G 31 23 base pairs nucleic acid single linear DNA(genomic) 19 GCAGCYTGTT TAGCAATAAA AGT 23 20 base pairs nucleic acidsingle linear DNA (genomic) 20 CAAAGGAAGA GTAGCTGTTA 20 25 base pairsnucleic acid single linear DNA (genomic) 21 CAATGTTAGC TTGGAAAATG TCACC25 22 base pairs nucleic acid single linear DNA (genomic) 22 GCTTAGTATCTACTTTAAAG AG 22 24 base pairs nucleic acid single linear DNA (genomic)23 GATACTATTT GATAAGTTCT CATC 24 24 base pairs nucleic acid singlelinear DNA (genomic) 24 CTTTTGGCCT TTTTGCGAAA TAAC 24 31 base pairsnucleic acid single linear DNA (genomic) 25 CTGGGTTTAA CGCTTTATCAGATTGATATT C 31 23 base pairs nucleic acid single linear DNA (genomic)26 ACTTTTATTG CTAAACARGC TGC 23 20 base pairs nucleic acid single linearDNA (genomic) 27 TAACAGCTAC TCTTCCTTTG 20 25 base pairs nucleic acidsingle linear DNA (genomic) 28 GGTGACATTT TCCAAGCTAA CATTG 25 21 basepairs nucleic acid single linear DNA (genomic) 29 CCAGTCCAAT GAACCTCTTAC 21 21 base pairs nucleic acid single linear DNA (genomic) 30AGGGAACAAA CCTTCCCAAC C 21 20 base pairs nucleic acid single linear DNA(genomic) 31 CARMTAKTAA MTAGGGATAG 20 22 base pairs nucleic acid singlelinear DNA (genomic) 32 AGYTTCTATC GAAGCTGGGR ST 22 1035 base pairsnucleic acid single linear DNA (genomic) 33 GGGTTAATTG GGTATTATTTTAAAGGGAAA GATTTTAATA ATCTGACTAT GTTTGCACCA 60 ACCATAAATA ATACGCTTATTTATGATCGG CAAACAGCAG ATACACTATT AAATAAGCAG 120 CAACAAGAGT TCAATTCTATTCGATGGATT GGTTTAATAC AAAGTAAAGA AACAGGTGAC 180 TTTACATTCC AATTATCAGATGATAAAAAT GCCATCATTG AAATAGATGG AAAAGTTGTT 240 TCTCGTAGAG GAGAAGATAAACAAACTATC CATTTAGAAA AAGGAAAGAT GGTTCCAATC 300 AAAATTGAGT ACCAGTCCAATGAACCTCTT ACTGTAGATA GTAAAGTATT TAACGATCTT 360 AAACTATTTA AAATAGATGGTCATAATCAA TCGCATCAAA TACAGCAAGA TGATTTGAAA 420 ATCCTGAATT TAATAAAAAGGAAACGAAAG AGCTTTTATC AAAAACAGCA AAAAGAACCT 480 TTTCTCTTCA AAACGGGGTTGAGAAGCGAT GAGGATGATG ATCTAGGATA CAGATGGTGA 540 TAGCATTCCT GGATAATTGGGAAATGAATG GATATACCAT TCAAACGAAA AATGGCAGTC 600 AAATGGGATG ATTCATTTGCAGAAAAAGGA TATACAAAAT TTGTTTCGAA TCCATATGAA 660 GCCCATACAG CAGGAGATCCTTATACCGAT TATGAAAAAG CAGCAAAAGA TATTCCTTTA 720 TCGAACGCAA AAGAAGCCTTTAATCCTCTT GTAGCTGCTT TTCCATCTGT CAATGTAGGA 780 TTAGAAAAAG TAGTAATTTCTAAAAATGAG GATATGAGTC AGGGTGTATC ATCCAGCACT 840 TCGAATAGTG CCTCTAATACAAATTCAATT GGTGTTACCG TAGATGCTGG TTGGGAAGGT 900 TTGTTCCCTA AATTTGGTATTTCAACTAAT TATCAAAACA CATGGACCAC TGCACAAGAA 960 TGGGGCTCTT CTAAAGAAGATTCTACCCAT ATAAATGGAG CACAATCAGC CTTTTTAAAT 1020 GCAAATGTAC GATAT 1035345 amino acids amino acid single linear protein 34 Gly Leu Ile Gly TyrTyr Phe Lys Gly Lys Asp Phe Asn Asn Leu Thr 1 5 10 15 Met Phe Ala ProThr Ile Asn Asn Thr Leu Ile Tyr Asp Arg Gln Thr 20 25 30 Ala Asp Thr LeuLeu Asn Lys Gln Gln Gln Glu Phe Asn Ser Ile Arg 35 40 45 Trp Ile Gly LeuIle Gln Ser Lys Glu Thr Gly Asp Phe Thr Phe Gln 50 55 60 Leu Ser Asp AspLys Asn Ala Ile Ile Glu Ile Asp Gly Lys Val Val 65 70 75 80 Ser Arg ArgGly Glu Asp Lys Gln Thr Ile His Leu Glu Lys Gly Lys 85 90 95 Met Val ProIle Lys Ile Glu Tyr Gln Ser Asn Glu Pro Leu Thr Val 100 105 110 Asp SerLys Val Phe Asn Asp Leu Lys Leu Phe Lys Ile Asp Gly His 115 120 125 AsnGln Ser His Gln Ile Gln Gln Asp Asp Leu Lys Ile Leu Asn Leu 130 135 140Ile Lys Arg Lys Arg Lys Ser Phe Tyr Gln Lys Gln Gln Lys Glu Pro 145 150155 160 Phe Leu Phe Lys Thr Gly Leu Arg Ser Asp Glu Asp Asp Asp Leu Gly165 170 175 Tyr Arg Trp Xaa Xaa His Ser Trp Ile Ile Gly Lys Xaa Met AspIle 180 185 190 Pro Phe Lys Arg Lys Met Ala Val Lys Trp Asp Asp Ser PheAla Glu 195 200 205 Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Tyr Glu AlaHis Thr Ala 210 215 220 Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala LysAsp Ile Pro Leu 225 230 235 240 Ser Asn Ala Lys Glu Ala Phe Asn Pro LeuVal Ala Ala Phe Pro Ser 245 250 255 Val Asn Val Gly Leu Glu Lys Val ValIle Ser Lys Asn Glu Asp Met 260 265 270 Ser Gln Gly Val Ser Ser Ser ThrSer Asn Ser Ala Ser Asn Thr Asn 275 280 285 Ser Ile Gly Val Thr Val AspAla Gly Trp Glu Gly Leu Phe Pro Lys 290 295 300 Phe Gly Ile Ser Thr AsnTyr Gln Asn Thr Trp Thr Thr Ala Gln Glu 305 310 315 320 Trp Gly Ser SerLys Glu Asp Ser Thr His Ile Asn Gly Ala Gln Ser 325 330 335 Ala Phe LeuAsn Ala Asn Val Arg Tyr 340 345 1037 base pairs nucleic acid singlelinear DNA (genomic) 35 GGGTTAATTG GGTATTATTT TAAAGGGAAA GATTTTAATAATCTGACTAT GTTTGCACCA 60 ACCATAAATA ATACGCTTAT TTATGATCGG CAAACAGCAGATACACTATT AAATAAGCAG 120 CAACAAGAGT TCAATTCTAT TCGATGGATT GGTTTAATACAAAGTAAAGA AACAGGTGAC 180 TTTACATTCC AATTATCAGA TGATAAAAAT GCCATCATTGAAATAGATGG AAAAGTTGTT 240 TCTCGTAGAG GAGAAGATAA ACAAACTATC CATTTAGAAAAAGGAAAGAT GGTTCCAATC 300 AAAATTGAGT ACCAGTCCAA TGAACCTCTT ACTGTAGATAGTAAAGTATT TAACGATCTT 360 AAACTATTTA AAATAGATGG TCATAATCAA TCGCATCAAATACAGCAAGA TGATTTGAAA 420 AATCCTGAAT TTAATAAAAA AGAAACGAAA GAGCTTTTATCAAAAACAGC AAAAAGRAAC 480 CTTTTCTCTT CAAACGRRGT KGAGAAGCGA TGAGGATGATRATCYTAGAT ACAGGTGGKG 540 ATAGCATTCC YKGATAATTG GGGAAATGAA WGGRTATACCATTCAACSGA AAAATGGSAG 600 TCAAATGGGA TGATTCATTT GCGGAAAAAG GATATACAAAATTTGTTTCG AATCCATATG 660 AAGCCCATAC AGCAGGAGAT CCTTATACCG ATTATGAAAAAGCAGCAAAA GATATTCCTT 720 TATCGAACGC AAAAGAAGCC TTTAATCCTC TTGTAGCTGCTTTTCCATCT GTCAATGTAG 780 GATTAGAAAA AGTAGTAATT TCTAAAAATG AGGATATGAGTCAGGGTGTA TCATCCAGCA 840 CTTCGAATAG TGCCTCTAAT ACAAATTCAA TTGGTGTTACCGTAGATGCT GGTTGGGAAG 900 GTTTGTTCCC TAAATTTGGT ATTTCAACTA ATTATCAAAACACATGGACC ACTGCACAAG 960 AATGGGGCTC TTCTAAAGAA GATTCTACCC ATATAAATGGAGCACAATCA GCCTTTTTAA 1020 ATGCAAATGT ACGATAT 1037 1048 base pairsnucleic acid single linear DNA (genomic) 36 TGGGTTAATT GGGTATTATTTTAAAGGGCA AGAGTTTAAT CATCTTACTT TGTTCGCACC 60 AACACGTGAT AATACCCTTATTTATGATCA ACAAACAGCG AATTCCTTAT TAGATACCAA 120 GCAACAAGAA TATCAATCTATTCGCTGGAT TGGTTTAATT CAAAGTAAAG AAACGGGTGA 180 TTTCACATTT AACTTATCAGATGATCAACA TGCAATTATA GAAATCGATG GCAAAATCAT 240 TTCGCATAAA GGACAGAATAAACAAGTTGT TCACTTAGAA AAAGGAAAGT TAGTCCCGAT 300 AAAAATTGAG TATCAATCAGATCAACTATT AAATAGGGAT AGTAACATCT TTAAAGAGTT 360 TAAATTATTC AAAGTAGATAGTCAGCAACA CGCTCACCAA GTTCAACTAG ACGAATTAAG 420 AAACCCTGCG TTTAATAAAAAGGAAACACA ACAATCTTAA GAAAAAGCAT CCAAAAACAA 480 TCTTTTTACA CCAGGGACATTAAAAGGAAG ATACTGATGA TGATGATAAG GATAACAGGA 540 TGGGAGATTC TATTCCTGGACCTTTTGGGG GAAGAAAATG GGTATACCAA TCCCAAAATA 600 AAATAGCTGG TCCAAGTGGGATGTTCATTC GCCGCGAAAG GGTATACAAA TTTGTTTCTT 660 AATCCACTTG ATAGTCATACAGTTGGAGAT CCCTATACGG ATTATGAAAA AGCAGCAAGA 720 GATTTAGACT TGGCCCAATGCAAAAGAAAC ATTTAACCCA TTAGTAGCTG CTTTTCCAAG 780 TGTGAATGTG AATTTGGAAAAAGTCATTTT ATCTAAAGAT GAAAATCTAT CCAATAGTGT 840 AGAGTCACAT TCCTCCACCAACTGGTCTTA TACGAATACA GAAGGAGCTT CTATCGAAGC 900 TGGGGCTAAA CCAGAGGGTCCTACTTTTGG AGTGAGTGCT ACTTATCAAC ACTCTGAAAC 960 AGTTGCAAAA GAATGGGGAACATCTACAGG AAATACCTCG CAATTTAATA CAGCTTCAGC 1020 AGGATATTTA AATGCAAATGTACGATAT 1048 1175 base pairs nucleic acid single linear DNA (genomic)37 ACCTCTAGAT GCANGCTCGA GCGGCCGCCA GTGTGATGGA TATCTGCAGA ATTCGGATTA 60CTTGGGTATT ATTTTAAAGG GAAAGAGTTT AATCATCTTA CTTTGTTCGC ACCAACACGT 120GATAATACCC TTATTTATGA TCAACAAACA GCGAATTCCT TATTAGATAC CAAACAACAA 180GAATATCAAT CTATTCGCTG GATTGGTTTG ATTCAAAGTA AAGAAACAGG TGATTTCACG 240TTTAACTTAT CTGATGATCA AAATGCAATT ATAGAAATAG ATGGCAAAAT CATTTCGCAT 300AAAGGACAGA ATAAACAAGT TGTTCACTTA GAAAAAGGAA AGTTAGTCCC GATAAAAATT 360GAGTATCAAT CAGATCAGAT ATTAACTAGG GATAGTAACA TCTTTAAAGA GTTCAATTAT 420TCAAAGTAGA TAGTCAAGCA ACACTCTCAC CAAAGTTCAA CTTAGGNCNG AATTAAGNAA 480CCCTNGGATT TTAANTTNAA AAAAAGGAAC CCNCANCATT CTTTAGGAAA AAGCAGCAAN 540AACCAAATCC TTTTTTACCA CAGGATATTG AAAAGGAGAT ACGGGNTNGA TGATGGATTG 600ATACCGGGAT ACCAGTTGGG GNTTCTANTC CCTGACCTTT GGGGAAAGAA AATNGGTATA 660CCNATCCCAA AANTTAAGCC AGCTGTCCAG GTGGGATGAT TCAATTCGCC CGCGAAAGGG 720TATACCAAAA TTTGTTTCTT AATCCACTTG AGAGTCATAC AGTTGGAGAT CCCTATACGG 780ATTATGAAAA AGCAGCAAGA GATTTAGACT TGGCCAATGC AAAAGAAACA TTTAACCCAT 840TAGTAGCTGC TTTTCCAAGT GTGAATGTGA ATTTGGAAAA AGTAATATTA TCCCCAGATG 900AGAATTTATC TAACAGTGTA GAATCTCATT CGTCTACAAA TTGGTCTTAT ACGAATACTG 960AAGGAGCTTC TATCGAAGCT GGGGGTGGTC CATTAGGTAT TTCATTTGGA GTGAGTGCTA 1020ATTATCAACA CTCTGAAACA GTTGCAAAAG AATGGGGAAC ATCTACAGGA AATACCTCGC 1080AATTTAATAC AGCTTCAGCA GGATATTTAA ATGCCAATGG TCGATNTAAG CCGAATNCCA 1140NCACACTGNC GGCCGTTAGT AGTGGCACCG AGCCC 1175 1030 base pairs nucleic acidsingle linear DNA (genomic) 38 GGRTTAMTTG GGTATTATTT TAAAGGGAAAGATTTTAATG ATCTTACTGT ATTTGCACCA 60 ACGCGTGGGA ATACTCTTGT ATATGATCAACAAACAGCAA ATACATTACT AAATCAAAAA 120 CAACAAGACT TTCAGTCTAT TCGTTGGGTTGGTTTAATTC AAAGTAAAGA AGCAGGCGAT 180 TTTACATTTA ACTTATCAGA TGATGAACATACGATGATAG AAATCGATGG GAAAGTTATT 240 TCTAATAAAG GGAAAGAAAA ACAAGTTGTCCATTTAGAAA AAGGACAGTT CGTTTCTATC 300 AAAATAGAAT ATCAAGCTGA TGAACCATTTAATGCGGATA GTCAAACCTT TAAAAATTTG 360 AAACTCYTTA AAGTAGATAC TAAGCAACAGTCCCAGCAAA TTCAACTAGA TGAATTAAGA 420 AACCCTGRAA TTTAATAAAA AAGAAACACAAGAATTTCTA ACAAAAGCAA CAAAAACAAA 480 CCTTATTACT CAAAAAGTGA AGAGTACTAGGGATGAAGAC ACGGATACAG ATGGAGATTC 540 TATTCCAGAC ATTTGGGAAG AAAATGGGTATACCATCCAA AATAAGATTG CCGTCAAATG 600 GGATGATTCA TTAGCAAGTA AAGGATATACGAAATTTGTT TCAAACCCAC TAGATACTCA 660 CACGGTTGGA GATCCTTATA CAGATTATGAAAAAGCAGCA AGGGATTTAG ATTTGTCAAA 720 TGCAAAAGAA ACATTTAACC CATTAGTTGCGGCTTTTCCA AGTGTGAATG TGAGTATGGA 780 AAAAGTGATA TTGTCTCCAG ATGAGAACTTATCAAATAGT ATCGAGTCTC ATTCATCTAC 840 GAATTGGTCG TATACGAATA CAGAAGGGGCTTCTATTGAA GCTGGTGGGG GAGCATTAGG 900 CCTATCTTTT GGTGTAAGTG CAAACTATCAACATTCTGAA ACAGTTGGGT ATGAATGGGG 960 AACATCTACG GGAAATACTT CGCAATTTAATACAGCTTCA GCGGGGTATT TAAATGCCAA 1020 TRTAMGATAT 1030 23 base pairsnucleic acid single linear DNA (genomic) 39 CACTCAAAAA ATGAAAAGGG AAA 2319 base pairs nucleic acid single linear DNA (genomic) 40 CCGGTTTTATTGATGCTAC 19 20 base pairs nucleic acid single linear DNA (genomic) 41AGAACAATTT TTAGATAGGG 20 20 base pairs nucleic acid single linear DNA(genomic) 42 TCCCTAAAGC ATCAGAAATA 20 1170 base pairs nucleic acidsingle linear DNA (genomic) 43 ATGAAGAAAC AAATAGCAAG CGTTGTAACTTGTACGCTAT TAGCCCCTAT GCTTTTTAAT 60 GGAGATATGA ACGCTGCTTA CGCAGCTAGTCAAACAAAAC AAACACCTGC AGCTCAGGTA 120 AACCAAGAGA AAGAAGTAGA TCGAAAAGGATTACTTGGCT ATTACTTTAA AGGGAAAGAT 180 TTTAATGATC TTACTGTATT TGCACCAACGCGTGGGAATA CTCTTGTATA TGATCAACAA 240 ACAGCAAATA CATTACTAAA TCAAAAACAACAAGACTTTC AGTCTATTCG TTGGGTTGGT 300 TTAATTCAAA GTAAAGAAGC AGGCGATTTTACATTTAACT TATCAGATGA TGAACATACG 360 ATGATAGAAA TCGATGGGAA AGTTATTTCTAATAAAGGGA AAGAAAAACA AGTTGTCCAT 420 TTAGAAAAAG GACAGTTCGT TTCTATAAAATGATTCAGCT GATGAACCAT TTAATGCGGT 480 AGTAAACCTT TAAAAATTTG AAACTCTTTAAAGTAGATAC TAAGCAACAG TCCCAGCAAA 540 TTCAACTAGA TGAATTAAGA AACCCTGAATTTAATAAAAA AGAAACACAA GAATTTCTAA 600 CAAAAGCAAC AAAAACAAAC CTTATTACTCAAAAAGTGAA GAGTACTAGG GATGAAGACA 660 CGGATACAGA TGGAGATTCT ATTCCAGACATTTGGGAAGA AAATGGGTAT ACCATCCAAA 720 ATAAATTGCC GTCAAATGGG ATGATTCATTAGCAAGTAAA GGATATACGA AATTTGTTTC 780 AAACCCACTA GATACTCACA CGGTTGGAGATCCTTATACA GATTATGAAA AAGCAGCAAG 840 GGATTTAGAT TTGTCAAATG CAAAAGAAACATTTAACCCA TTAGTTGCGG CTTTTCCAAG 900 TGTAATTGAG TATGGAAAAA GGATTTGTTCCAGATGAGAA CTTATCAAAT AGTATCGAGT 960 TCATTCATTC CTACAATTGG TCGATACGAATACAGAAGGG GCTTCTATTG AAGCTGGTGG 1020 GGGAGCATTA GGCCTATCTT TTGGTGTAAGTGCAAACTAT CAACATTCTG AAACAGTTGG 1080 GTATGAATGG GGAACATCTA CGGGAAATACTTCGCAATTT AATACAGCTT CAGCGGGGTA 1140 TTTAAATGCG AATGTTGCTA CAATAACGTG1170 348 amino acids amino acid single linear protein 44 Met Lys Lys GlnIle Ala Ser Val Val Thr Cys Thr Leu Leu Ala Pro 1 5 10 15 Met Leu PheAsn Gly Asp Met Asn Ala Ala Tyr Ala Ala Ser Gln Thr 20 25 30 Lys Gln ThrPro Ala Ala Gln Val Asn Gln Glu Lys Glu Val Asp Arg 35 40 45 Lys Gly LeuLeu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asp Leu 50 55 60 Thr Val PheAla Pro Thr Arg Gly Asn Thr Leu Val Tyr Asp Gln Gln 65 70 75 80 Thr AlaAsn Thr Leu Leu Asn Gln Lys Gln Gln Asp Phe Gln Ser Ile 85 90 95 Arg TrpVal Gly Leu Ile Gln Ser Lys Glu Ala Gly Asp Phe Thr Phe 100 105 110 AsnLeu Ser Asp Asp Glu His Thr Met Ile Glu Ile Asp Gly Lys Val 115 120 125Ile Ser Asn Lys Gly Lys Glu Lys Gln Val Val His Leu Glu Lys Gly 130 135140 Gln Phe Val Ser Xaa Lys Xaa Xaa Xaa Xaa Ala Asp Glu Pro Phe Asn 145150 155 160 Ala Xaa Ser Xaa Thr Phe Lys Asn Leu Lys Leu Phe Lys Val AspThr 165 170 175 Lys Gln Gln Ser Gln Gln Ile Gln Leu Asp Glu Leu Arg AsnPro Glu 180 185 190 Phe Asn Lys Lys Glu Thr Gln Glu Phe Leu Thr Lys AlaThr Lys Thr 195 200 205 Asn Leu Ile Thr Gln Lys Val Lys Ser Thr Arg AspGlu Asp Thr Asp 210 215 220 Thr Asp Gly Asp Ser Ile Pro Asp Ile Trp GluGlu Asn Gly Tyr Thr 225 230 235 240 Ile Gln Asn Xaa Ile Ala Val Lys TrpAsp Asp Ser Leu Ala Ser Lys 245 250 255 Gly Tyr Thr Lys Phe Val Ser AsnPro Leu Asp Thr His Thr Val Gly 260 265 270 Asp Pro Tyr Thr Asp Tyr GluLys Ala Ala Arg Asp Leu Asp Leu Ser 275 280 285 Asn Ala Lys Glu Thr PheAsn Pro Leu Val Ala Ala Phe Pro Ser Val 290 295 300 Asn Xaa Ser Met GluLys Xaa Ile Leu Xaa Pro Asp Glu Asn Leu Ser 305 310 315 320 Asn Ser IleGlu Xaa His Ser Phe Leu Xaa Ile Gly Arg Ile Arg Ile 325 330 335 Gln LysGly Leu Leu Leu Lys Leu Val Gly Glu His 340 345 3 base pairs nucleicacid single linear DNA (genomic) 45 ATG 3 1 amino acids amino acidsingle linear protein 46 Met 1 2583 base pairs nucleic acid singlelinear DNA (genomic) 47 ATGACATATA TGAAAAAAAA GTTAGTTAGT GTTGTAACTTGCACGTTATT GGCTCCGATA 60 TTTTTGACTG GAAATGTACA TCCTGTTAAT GCAGACAGTAAAAAAAGTCA GCCTTCTACA 120 GCGCAGGAAA AACAAGAAAA GCCGGTTGAT CGAAAAGGGTTACTCGGCTA TTTTTTTAAA 180 GGGAAAGAGT TTAATCATCT TACTTTGTTC GCACCAACACGTGATAATAC CCTTATTTAT 240 GATCAACAAA CAGCGAATTC CTTATTAGAT ACCAAACAACAAGAATATCA ATCTATTCGC 300 TGGATTGGTT TGATTCAAAG TAAAGAAACA GGTGATTTCACGTTTAACTT ATCTGATGAT 360 CAAAATGCAA TTATAGAAAT AGATGGCAAA ATCATTTCGCATAAAGGACA GAATAAACAA 420 GTTGTTCACT TAGAAAAAGG AAAGTTAGTC CCGATAAAAATTGAGTATCA ATCAGATCAG 480 ATATTAACTA GGGATAGTAA CATCTTTAAA GAGTTTCAATTATTCAAAGT AGATAGTCAG 540 CAACACTCTC ACCAAGTTCA ACTAGACGAA TTAAGAAACCCTGATTTTAA TAAAAAAGAA 600 ACACAACAAT TCTTAGAAAA AGCAGCAAAA ACAAATCTTTTTACACAGAA TATGAAAAGA 660 GATACGGATG ATGATGATGA TACGGATACA GATGGAGATTCTATTCCTGA CCTTTGGGAA 720 GAAAATGGGT ATACCATCCA AAATAAAGTA GCTGTCAAGTGGGATGATTC ATTCGCCGCG 780 AAAGGGTATA CAAAATTTGT TTCTAATCCA CTTGAGAGTCATACAGTTGG AGATCCCTAT 840 ACGGATTATG AAAAAGCAGC AAGAGATTTA GACTTGGCCAATGCAAAAGA AACATTTAAC 900 CCATTAGTAG CTGCTTTTCC AAGTGTGAAT GTGAATTTGGAAAAAGTAAT ATTATCCCCA 960 GATGAGAATT TATCTAACAG TGTAGAATCT CATTCGTCTACAAATTGGTC TTATACGAAT 1020 ACTGAAGGAG CTTCTATCGA AGCTGGGGGT GGTCCATTAGGTATTTCATT TGGAGTGAGT 1080 GCTAATTATC AACACTCTGA AACAGTTGCA AAAGAATGGGGAACATCTAC AGGAAATACC 1140 TCGCAATTTA ATACAGCTTC AGCAGGATAT TTGAATGCGAATGTTCGATA CAATAATGTG 1200 GGAACAGGTG CGATTTATGA GGTGAAACCT ACAACAAGTTTTGTATTAGA TAAAGATACT 1260 GTAGCAACAA TTACCGCAAA ATCGAATTCG ACAGCTTTAAGTATATCTCC AGGAGAAAGT 1320 TATCCCAAAA AAGGACAAAA TGGAATTGCA ATTAATACAATGGATGATTT TAATTCCCAT 1380 CCGATTACAT TAAATAAACA ACAATTAGAT CAACTATTAAATAATAAACC TCTTATGTTA 1440 GAAACAAATC AGGCAGATGG TGTTTATAAA ATAAAGGATACAAGCGGTAA TATTGTGACT 1500 GGTGGAGAAT GGAACGGTGT TATCCAACAA ATTCAAGCAAAAACAGCCTC TATTATCGTT 1560 GATACGGGAG AAAGTGTTTC AGAAAAGCGT GTCGCAGCAAAAGATTATGA TAATCCTGAG 1620 GATAAAACAC CTTCTTTATC TTTAAAAGAG GCACTTAAACTTGGATATCC AGAAGAAATT 1680 AAAGAAAAAG ATGGATTGTT GTACTATAAG GACAAGCCAATTTACGAATC TAGTGTTATG 1740 ACTTATCTAG ATGAGAATAC AGCCAAGGAA GTGGAAAAACAATTACAGGA TACAACCGGA 1800 ATATATAAAG ATATCAATCA TTTATATGAT GTGAAATTAACACCTACAAT GAATTTTACG 1860 ATTAAATTAG CTTCCTTATA TGATGGAGCT GAAAATAATGATGTGAAGAA TGGTCCTATA 1920 GGACATTGGT ATTATACCTA TAATACAGGG GGAGGAAATACTGGAAAACA CCAATATAGG 1980 TCTGCTAATC CCAGTGCAAA TGTAGTTTTA TCTTCTGAAGCGAAAAGTAA GTTAGATAAA 2040 AATACAAATT ACTACCTTAG TATGTATATG AAAGCTGAGTCTGATACAGA GCCTACAATA 2100 GAAGTAAGTG GTGAGAATTC TACGATAACG AGTAAAAAGGTAAAACTAAA CAGTGAGGGC 2160 TATCAAAGAG TAGATATTTT AGTGCCGAAT TCTGAAAGAAATCCAATAAA TCAAATATAT 2220 GTAAGAGGAA ATAATACAAC AAATGTATAC TGGGATGATGTTTCAATTAC AAATATTTCA 2280 GCTATAAACC CAAAAACTTT AACAGATGAA GAAATTAAAGAAATATATAA AGATTTTAGT 2340 GAGTCTAAAG ACTGGCCTTG GTTCAATGAT GTTACGTTTAAAAATATTAA ACCATTAGAG 2400 AATTATGTAA AACAATATAG AGTTGATTTC TGGAATACTAATAGTGATAG ATCATTTAAT 2460 AGGATTAAGG ACAGTTACCC AGTTAATGAA GATGGAAGTGTTAAAGTCAA CATGACAGAA 2520 TATAATGAAG GATATCCACT TAGAATTGAA TCCGCCTACCATTTAAATAT TTCAGATCTA 2580 TAA 2583 860 amino acids amino acid singlelinear protein 48 Met Thr Tyr Met Lys Lys Lys Leu Val Ser Val Val ThrCys Thr Leu 1 5 10 15 Leu Ala Pro Ile Phe Leu Thr Gly Asn Val His ProVal Asn Ala Asp 20 25 30 Ser Lys Lys Ser Gln Pro Ser Thr Ala Gln Glu LysGln Glu Lys Pro 35 40 45 Val Asp Arg Lys Gly Leu Leu Gly Tyr Phe Phe LysGly Lys Glu Phe 50 55 60 Asn His Leu Thr Leu Phe Ala Pro Thr Arg Asp AsnThr Leu Ile Tyr 65 70 75 80 Asp Gln Gln Thr Ala Asn Ser Leu Leu Asp ThrLys Gln Gln Glu Tyr 85 90 95 Gln Ser Ile Arg Trp Ile Gly Leu Ile Gln SerLys Glu Thr Gly Asp 100 105 110 Phe Thr Phe Asn Leu Ser Asp Asp Gln AsnAla Ile Ile Glu Ile Asp 115 120 125 Gly Lys Ile Ile Ser His Lys Gly GlnAsn Lys Gln Val Val His Leu 130 135 140 Glu Lys Gly Lys Leu Val Pro IleLys Ile Glu Tyr Gln Ser Asp Gln 145 150 155 160 Ile Leu Thr Arg Asp SerAsn Ile Phe Lys Glu Phe Gln Leu Phe Lys 165 170 175 Val Asp Ser Gln GlnHis Ser His Gln Val Gln Leu Asp Glu Leu Arg 180 185 190 Asn Pro Asp PheAsn Lys Lys Glu Thr Gln Gln Phe Leu Glu Lys Ala 195 200 205 Ala Lys ThrAsn Leu Phe Thr Gln Asn Met Lys Arg Asp Thr Asp Asp 210 215 220 Asp AspAsp Thr Asp Thr Asp Gly Asp Ser Ile Pro Asp Leu Trp Glu 225 230 235 240Glu Asn Gly Tyr Thr Ile Gln Asn Lys Val Ala Val Lys Trp Asp Asp 245 250255 Ser Phe Ala Ala Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu 260265 270 Ser His Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg275 280 285 Asp Leu Asp Leu Ala Asn Ala Lys Glu Thr Phe Asn Pro Leu ValAla 290 295 300 Ala Phe Pro Ser Val Asn Val Asn Leu Glu Lys Val Ile LeuSer Pro 305 310 315 320 Asp Glu Asn Leu Ser Asn Ser Val Glu Ser His SerSer Thr Asn Trp 325 330 335 Ser Tyr Thr Asn Thr Glu Gly Ala Ser Ile GluAla Gly Gly Gly Pro 340 345 350 Leu Gly Ile Ser Phe Gly Val Ser Ala AsnTyr Gln His Ser Glu Thr 355 360 365 Val Ala Lys Glu Trp Gly Thr Ser ThrGly Asn Thr Ser Gln Phe Asn 370 375 380 Thr Ala Ser Ala Gly Tyr Leu AsnAla Asn Val Arg Tyr Asn Asn Val 385 390 395 400 Gly Thr Gly Ala Ile TyrGlu Val Lys Pro Thr Thr Ser Phe Val Leu 405 410 415 Asp Lys Asp Thr ValAla Thr Ile Thr Ala Lys Ser Asn Ser Thr Ala 420 425 430 Leu Ser Ile SerPro Gly Glu Ser Tyr Pro Lys Lys Gly Gln Asn Gly 435 440 445 Ile Ala IleAsn Thr Met Asp Asp Phe Asn Ser His Pro Ile Thr Leu 450 455 460 Asn LysGln Gln Leu Asp Gln Leu Leu Asn Asn Lys Pro Leu Met Leu 465 470 475 480Glu Thr Asn Gln Ala Asp Gly Val Tyr Lys Ile Lys Asp Thr Ser Gly 485 490495 Asn Ile Val Thr Gly Gly Glu Trp Asn Gly Val Ile Gln Gln Ile Gln 500505 510 Ala Lys Thr Ala Ser Ile Ile Val Asp Thr Gly Glu Ser Val Ser Glu515 520 525 Lys Arg Val Ala Ala Lys Asp Tyr Asp Asn Pro Glu Asp Lys ThrPro 530 535 540 Ser Leu Ser Leu Lys Glu Ala Leu Lys Leu Gly Tyr Pro GluGlu Ile 545 550 555 560 Lys Glu Lys Asp Gly Leu Leu Tyr Tyr Lys Asp LysPro Ile Tyr Glu 565 570 575 Ser Ser Val Met Thr Tyr Leu Asp Glu Asn ThrAla Lys Glu Val Glu 580 585 590 Lys Gln Leu Gln Asp Thr Thr Gly Ile TyrLys Asp Ile Asn His Leu 595 600 605 Tyr Asp Val Lys Leu Thr Pro Thr MetAsn Phe Thr Ile Lys Leu Ala 610 615 620 Ser Leu Tyr Asp Gly Ala Glu AsnAsn Asp Val Lys Asn Gly Pro Ile 625 630 635 640 Gly His Trp Tyr Tyr ThrTyr Asn Thr Gly Gly Gly Asn Thr Gly Lys 645 650 655 His Gln Tyr Arg SerAla Asn Pro Ser Ala Asn Val Val Leu Ser Ser 660 665 670 Glu Ala Lys SerLys Leu Asp Lys Asn Thr Asn Tyr Tyr Leu Ser Met 675 680 685 Tyr Met LysAla Glu Ser Asp Thr Glu Pro Thr Ile Glu Val Ser Gly 690 695 700 Glu AsnSer Thr Ile Thr Ser Lys Lys Val Lys Leu Asn Ser Glu Gly 705 710 715 720Tyr Gln Arg Val Asp Ile Leu Val Pro Asn Ser Glu Arg Asn Pro Ile 725 730735 Asn Gln Ile Tyr Val Arg Gly Asn Asn Thr Thr Asn Val Tyr Trp Asp 740745 750 Asp Val Ser Ile Thr Asn Ile Ser Ala Ile Asn Pro Lys Thr Leu Thr755 760 765 Asp Glu Glu Ile Lys Glu Ile Tyr Lys Asp Phe Ser Glu Ser LysAsp 770 775 780 Trp Pro Trp Phe Asn Asp Val Thr Phe Lys Asn Ile Lys ProLeu Glu 785 790 795 800 Asn Tyr Val Lys Gln Tyr Arg Val Asp Phe Trp AsnThr Asn Ser Asp 805 810 815 Arg Ser Phe Asn Arg Ile Lys Asp Ser Tyr ProVal Asn Glu Asp Gly 820 825 830 Ser Val Lys Val Asn Met Thr Glu Tyr AsnGlu Gly Tyr Pro Leu Arg 835 840 845 Ile Glu Ser Ala Tyr His Leu Asn IleSer Asp Leu 850 855 860 1356 base pairs nucleic acid single linear DNA(genomic) 49 ATGGTATCCA AAAAGTTACA ATTAGTCACA AAAACTTTAG TGTTTAGTACAGTTTTGTCA 60 ATACCGTTAT TAAATAATAG TGAGATAAAA GCGGAACAAT TAAATATGAATTCTCAAATT 120 AAATATCCTA ACTTCCAAAA TATAAATATC GCTGATAAGC CAGTAGATTTTAAAGAGGAT 180 AAAGAAAAAG CACGAGAATG GGGAAAAGAA AAAGAAAAAG AGTGGAAACTAACTGCTACT 240 GAAAAAGGGA AAATTAATGA TTTTTTAGAT GATAAAGATG GATTAAAAACAAAATACAAA 300 GAAATTAATT TTTCTAAGAA TTTTGAATAT GAAACAGAGT TAAAACAGCTTGAAAAAATT 360 AATAGCATGC TAGATAAAGC AAATCTAACA AATTCAATTG TCACGTATAAAAACGTTGAG 420 CCTACAACAA TAGGATTCAA TCACTCTTTG ACTGATGGGA ATCAAATTAATTCCGAAGCT 480 CAACAGAAGT TCAAGGAACA GTTTTTAGGA AATGATATTA AATTTGATAGTTATTTGGAT 540 ATGCACTTAA CTGAACAAAA TGTTTCCGGT AAAGAAAGGG TTATTTTAAAAGTTACAGTA 600 CTTAGTGGGA AAGGTTCTAC TCCAACAAAA GCAGGTGTTG TTTTAAATAATAAAGAATAC 660 AAAATGTTGA TTGATAATGG ATATATACTA CATGTAGAAA ACATAACGAAAGTTGTAAAA 720 AAAGGACAGG AATGTTTACA AGTTGAAGGA ACGTTAAAAA AGAGCTTGGACTTTAAAAAT 780 GATAGTGACG GTAAGGGAGA TTCCTGGGGA AAGAAAAATT ACAAGGAATGGTCTGATTCT 840 TTAACAAATG ATCAGAGAAA AGACTTAAAT GATTATGGTG CGCGAGGTTATACCGAAATA 900 AATAAATATT TACGTGAAGG GGGTACCGGA AATACAGAGT TGGAGGAAAAAATTAAAAAT 960 ATTTCTGACG CACTAGAAAA GAATCCTATC CCTGAAAACA TTACTGTTTATAGATATTGC 1020 GGAATGGCGG AATTTGGTTA TCCAATTCAA CCCGAGGCTC CCTCCGTACAAGATTTTGAA 1080 GAGAAATTTT TGGATAAAAT TAAGGAAGAA AAAGGATATA TGAGTACGAGCTTATCAAGT 1140 GATGCGACTT CTTTTGGCGC AAGAAAAATT ATCTTAAGAT TGCAGATACCAAAAGGAAGT 1200 TCAGGAGCAT ATGTAGCTGG TTTAGATGGA TTTAAACCAG CAGAGAAGGAGATTCTTATT 1260 GATAAGGGAA GCAAGTATCA TATTGATAAA GTAACAGAAG TAGTTGTGAAAGGTATTAGA 1320 AAACTCGTAG TAGATGCGAC ATTATTATTA AAATAA 1356 451 aminoacids amino acid single linear DNA (genomic) 50 Met Val Ser Lys Lys LeuGln Leu Val Thr Lys Thr Leu Val Phe Ser 1 5 10 15 Thr Val Leu Ser IlePro Leu Leu Asn Asn Ser Glu Ile Lys Ala Glu 20 25 30 Gln Leu Asn Met AsnSer Gln Ile Lys Tyr Pro Asn Phe Gln Asn Ile 35 40 45 Asn Ile Ala Asp LysPro Val Asp Phe Lys Glu Asp Lys Glu Lys Ala 50 55 60 Arg Glu Trp Gly LysGlu Lys Glu Lys Glu Trp Lys Leu Thr Ala Thr 65 70 75 80 Glu Lys Gly LysIle Asn Asp Phe Leu Asp Asp Lys Asp Gly Leu Lys 85 90 95 Thr Lys Tyr LysGlu Ile Asn Phe Ser Lys Asn Phe Glu Tyr Glu Thr 100 105 110 Glu Leu LysGln Leu Glu Lys Ile Asn Ser Met Leu Asp Lys Ala Asn 115 120 125 Leu ThrAsn Ser Ile Val Thr Tyr Lys Asn Val Glu Pro Thr Thr Ile 130 135 140 GlyPhe Asn His Ser Leu Thr Asp Gly Asn Gln Ile Asn Ser Glu Ala 145 150 155160 Gln Gln Lys Phe Lys Glu Gln Phe Leu Gly Asn Asp Ile Lys Phe Asp 165170 175 Ser Tyr Leu Asp Met His Leu Thr Glu Gln Asn Val Ser Gly Lys Glu180 185 190 Arg Val Ile Leu Lys Val Thr Val Leu Ser Gly Lys Gly Ser ThrPro 195 200 205 Thr Lys Ala Gly Val Val Leu Asn Asn Lys Glu Tyr Lys MetLeu Ile 210 215 220 Asp Asn Gly Tyr Ile Leu His Val Glu Asn Ile Thr LysVal Val Lys 225 230 235 240 Lys Gly Gln Glu Cys Leu Gln Val Glu Gly ThrLeu Lys Lys Ser Leu 245 250 255 Asp Phe Lys Asn Asp Ser Asp Gly Lys GlyAsp Ser Trp Gly Lys Lys 260 265 270 Asn Tyr Lys Glu Trp Ser Asp Ser LeuThr Asn Asp Gln Arg Lys Asp 275 280 285 Leu Asn Asp Tyr Gly Ala Arg GlyTyr Thr Glu Ile Asn Lys Tyr Leu 290 295 300 Arg Glu Gly Gly Thr Gly AsnThr Glu Leu Glu Glu Lys Ile Lys Asn 305 310 315 320 Ile Ser Asp Ala LeuGlu Lys Asn Pro Ile Pro Glu Asn Ile Thr Val 325 330 335 Tyr Arg Tyr CysGly Met Ala Glu Phe Gly Tyr Pro Ile Gln Pro Glu 340 345 350 Ala Pro SerVal Gln Asp Phe Glu Glu Lys Phe Leu Asp Lys Ile Lys 355 360 365 Glu GluLys Gly Tyr Met Ser Thr Ser Leu Ser Ser Asp Ala Thr Ser 370 375 380 PheGly Ala Arg Lys Ile Ile Leu Arg Leu Gln Ile Pro Lys Gly Ser 385 390 395400 Ser Gly Ala Tyr Val Ala Gly Leu Asp Gly Phe Lys Pro Ala Glu Lys 405410 415 Glu Ile Leu Ile Asp Lys Gly Ser Lys Tyr His Ile Asp Lys Val Thr420 425 430 Glu Val Val Val Lys Gly Ile Arg Lys Leu Val Val Asp Ala ThrLeu 435 440 445 Leu Leu Lys 450 47 base pairs nucleic acid single linearDNA (genomic) 51 GCTCTAGAAG GAGGTAACTT ATGAACAAGA ATAATACTAA ATTAAGC 4727 base pairs nucleic acid single linear DNA (genomic) 52 GGGGTACCTTACTTAATAGA GACATCG 27 2364 base pairs nucleic acid single linear DNA(genomic) 53 ATGAATATGA ATAATACTAA ATTAAACGCA AGGGCCCTAC CGAGTTTTATTGATTATTTT 60 AATGGCATTT ATGGATTTGC CACTGGTATC AAAGACATTA TGAATATGATTTTTAAAACG 120 GATACAGGTG GTAATCTAAC CTTAGACGAA ATCCTAAAGA ATCAGCAGTTACTAAATGAG 180 ATTTCTGGTA AATTGGATGG GGTAAATGGG AGCTTAAATG ATCTTATCGCACAGGGAAAC 240 TTAAATACAG AATTATCTAA GGAAATCTTA AAAATTGCAA ATGAACAGAATCAAGTCTTA 300 AATGATGTTA ATAACAAACT CGATGCGATA AATACGATGC TTCATATATATCTACCTAAA 360 ATCACATCTA TGTTAAGTGA TGTAATGAAG CAAAATTATG CGCTAAGTCTGCAAGTAGAA 420 TACTTAAGTA AACAATTGAA AGAAATTTCT GATAAATTAG ATGTTATTAACGTAAATGTT 480 CTTATTAACT CTACACTTAC TGAAATTACA CCTGCATATC AACGGATTAAATATGTAAAT 540 GAAAAATTTG AAGAATTAAC TTTTGCTACA GAAACCACTT TAAAAGTAAAAAAGGATAGC 600 TCGCCTGCTG ATATTCTTGA CGAGTTAACT GAATTAACTG AACTAGCGAAAAGTGTTACA 660 AAAAATGACG TGGATGGTTT TGAATTTTAC CTTAATACAT TCCACGATGTAATGGTAGGA 720 AATAATTTAT TCGGGCGTTC AGCTTTAAAA ACTGCTTCAG AATTAATTGCTAAAGAAAAT 780 GTGAAAACAA GTGGCAGTGA AGTAGGAAAT GTTTATAATT TCTTAATTGTATTAACAGCT 840 CTACAAGCAA AAGCTTTTCT TACTTTAACA ACATGCCGAA AATTATTAGGCTTAGCAGAT 900 ATTGATTATA CATCTATTAT GAATGAACAT TTAAATAAGG AAAAAGAGGAATTTAGAGTA 960 AACATCCTTC CTACACTTTC TAATACTTTT TCTAATCCTA ATTATGCAAAAGTTAAAGGA 1020 AGTGATGAAG ATGCAAAGAT GATTGTGGAA GCTAAACCAG GACATGCATTGGTTGGGTTT 1080 GAAATTAGTA ATGATTCAAT GACAGTATTA AAAGTATATG AAGCTAAGCTAAAACAAAAT 1140 TACCAAGTTG ATAAGGATTC CTTATCGGAA GTCATTTATA GTGATATGGATAAATTATTG 1200 TGCCCAGATC AATCTGAACA AATTTATTAT ACAAATAATA TAGTATTTCCAAATGAATAT 1260 GTAATTACTA AAATTGATTT TACTAAGAAA ATGAAAACTT TAAGATATGAGGTAACAGCT 1320 AATTCTTACG ATTCTTCTAC AGGAGAAATT GACTTAAATA AGAAGAAAGTAGAATCAAGT 1380 GAAGCGGAGT ATAGGACGTT AAGTGCTAAT AATGATGGAG TATATATGCCGTTAGGTGTC 1440 ATCAGTGAAA CATTTTTGAC TCCAATTAAT GGATTTGGCC TCCAAGCTGATGAAAATTCA 1500 AGATTAATTA CTTTAACATG TAAATCATAT TTAAGGGAAC TACTACTAGCGACAGACTTA 1560 AGCAATAAAG AAACTAAATT GATTGTCCCG CCTATTAGTT TTATTAGTAATATTGTAGAA 1620 AATGGGAACT TAGAGGGAGA AAACTTAGAG CCGTGGATAG CAAATAACAAAAATGCGTAT 1680 GTAGATCATA CAGGTGGTAT AAATGGAACT AAAGTTTTAT ATGTTCATAAGGATGGTGAG 1740 TTTTCACAAT TTGTTGGAGG TAAGTTAAAA TCGAAAACAG AATATGTAATTCAATATATT 1800 GTAAAGGGAA AAGCTTCTAT TTATTTAAAA GATAAAAAAA ATGAGAATTCCATTTATGAA 1860 GAAATAAATA ATGATTTAGA AGGTTTTCAA ACTGTTACTA AACGTTTTATTACAGGAACG 1920 GATTCTTCAG GGATTCATTT AATTTTTACC AGTCAAAATG GCGAGGGAGCATTTGGAGGA 1980 AACTTTATTA TCTCAGAAAT TAGGACATCC GAAGAGTTAT TAAGTCCAGAATTGATTATG 2040 TCGGATGCTT GGGTTGGATC CCAGGGAACT TGGATCTCAG GAAATTCTCTCACTATTAAT 2100 AGTAATGTAA ATGGAACCTT TCGACAAAAT CTTCCGTTAG AAAGTTATTCAACCTATAGT 2160 ATGAACTTTA CTGTGAATGG ATTTGGCAAG GTGACAGTAA GAAATTCTCGTGAAGTATTA 2220 TTTGAAAAAA GTTATCCGCA GCTTTCACCT AAAGATATTT CTGAAAAATTTACAACTGCA 2280 GCCAATAATA CCGGATTATA TGTAGAGCTT TCTCGCTCAA CGTCGGGTGGTGCAATAAAT 2340 TTCCGAGATT TTTCAATTAA GTAA 2364 787 amino acids aminoacid single linear protein 54 Met Asn Met Asn Asn Thr Lys Leu Asn AlaArg Ala Leu Pro Ser Phe 1 5 10 15 Ile Asp Tyr Phe Asn Gly Ile Tyr GlyPhe Ala Thr Gly Ile Lys Asp 20 25 30 Ile Met Asn Met Ile Phe Lys Thr AspThr Gly Gly Asn Leu Thr Leu 35 40 45 Asp Glu Ile Leu Lys Asn Gln Gln LeuLeu Asn Glu Ile Ser Gly Lys 50 55 60 Leu Asp Gly Val Asn Gly Ser Leu AsnAsp Leu Ile Ala Gln Gly Asn 65 70 75 80 Leu Asn Thr Glu Leu Ser Lys GluIle Leu Lys Ile Ala Asn Glu Gln 85 90 95 Asn Gln Val Leu Asn Asp Val AsnAsn Lys Leu Asp Ala Ile Asn Thr 100 105 110 Met Leu His Ile Tyr Leu ProLys Ile Thr Ser Met Leu Ser Asp Val 115 120 125 Met Lys Gln Asn Tyr AlaLeu Ser Leu Gln Val Glu Tyr Leu Ser Lys 130 135 140 Gln Leu Lys Glu IleSer Asp Lys Leu Asp Val Ile Asn Val Asn Val 145 150 155 160 Leu Ile AsnSer Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile 165 170 175 Lys TyrVal Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr 180 185 190 ThrLeu Lys Val Lys Lys Asp Ser Ser Pro Ala Asp Ile Leu Asp Glu 195 200 205Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val 210 215220 Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly 225230 235 240 Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu LeuIle 245 250 255 Ala Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly AsnVal Tyr 260 265 270 Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Lys AlaPhe Leu Thr 275 280 285 Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala AspIle Asp Tyr Thr 290 295 300 Ser Ile Met Asn Glu His Leu Asn Lys Glu LysGlu Glu Phe Arg Val 305 310 315 320 Asn Ile Leu Pro Thr Leu Ser Asn ThrPhe Ser Asn Pro Asn Tyr Ala 325 330 335 Lys Val Lys Gly Ser Asp Glu AspAla Lys Met Ile Val Glu Ala Lys 340 345 350 Pro Gly His Ala Leu Val GlyPhe Glu Ile Ser Asn Asp Ser Met Thr 355 360 365 Val Leu Lys Val Tyr GluAla Lys Leu Lys Gln Asn Tyr Gln Val Asp 370 375 380 Lys Asp Ser Leu SerGlu Val Ile Tyr Ser Asp Met Asp Lys Leu Leu 385 390 395 400 Cys Pro AspGln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe 405 410 415 Pro AsnGlu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys 420 425 430 ThrLeu Arg Tyr Glu Val Thr Ala Asn Ser Tyr Asp Ser Ser Thr Gly 435 440 445Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr 450 455460 Arg Thr Leu Ser Ala Asn Asn Asp Gly Val Tyr Met Pro Leu Gly Val 465470 475 480 Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu GlnAla 485 490 495 Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser TyrLeu Arg 500 505 510 Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu ThrLys Leu Ile 515 520 525 Val Pro Pro Ile Ser Phe Ile Ser Asn Ile Val GluAsn Gly Asn Leu 530 535 540 Glu Gly Glu Asn Leu Glu Pro Trp Ile Ala AsnAsn Lys Asn Ala Tyr 545 550 555 560 Val Asp His Thr Gly Gly Ile Asn GlyThr Lys Val Leu Tyr Val His 565 570 575 Lys Asp Gly Glu Phe Ser Gln PheVal Gly Gly Lys Leu Lys Ser Lys 580 585 590 Thr Glu Tyr Val Ile Gln TyrIle Val Lys Gly Lys Ala Ser Ile Tyr 595 600 605 Leu Lys Asp Lys Lys AsnGlu Asn Ser Ile Tyr Glu Glu Ile Asn Asn 610 615 620 Asp Leu Glu Gly PheGln Thr Val Thr Lys Arg Phe Ile Thr Gly Thr 625 630 635 640 Asp Ser SerGly Ile His Leu Ile Phe Thr Ser Gln Asn Gly Glu Gly 645 650 655 Ala PheGly Gly Asn Phe Ile Ile Ser Glu Ile Arg Thr Ser Glu Glu 660 665 670 LeuLeu Ser Pro Glu Leu Ile Met Ser Asp Ala Trp Val Gly Ser Gln 675 680 685Gly Thr Trp Ile Ser Gly Asn Ser Leu Thr Ile Asn Ser Asn Val Asn 690 695700 Gly Thr Phe Arg Gln Asn Leu Pro Leu Glu Ser Tyr Ser Thr Tyr Ser 705710 715 720 Met Asn Phe Thr Val Asn Gly Phe Gly Lys Val Thr Val Arg AsnSer 725 730 735 Arg Glu Val Leu Phe Glu Lys Ser Tyr Pro Gln Leu Ser ProLys Asp 740 745 750 Ile Ser Glu Lys Phe Thr Thr Ala Ala Asn Asn Thr GlyLeu Tyr Val 755 760 765 Glu Leu Ser Arg Ser Thr Ser Gly Gly Ala Ile AsnPhe Arg Asp Phe 770 775 780 Ser Ile Lys 785

What is claimed is:
 1. An isolated polynucleotide that encodes apesticidally active protein, wherein said protein has at least 95%identity with the amino acid sequence of SEQ ID NO:54.
 2. Thepolynucleotide according to claim 1, wherein said protein comprises theamino acid sequence of SEQ ID NO:54.
 3. The polynucleotide according toclaim 1, wherein said polynucleotide comprises the nucleotide sequenceof SEQ ID NO:53.
 4. A host cell comprising an isolated polynucleotidethat encodes a pesticidally active protein, wherein said protein has atleast 95% identity with the amino acid sequence of SEQ ID NO:54, andwherein said cell is selected from the group consisting of a plant celland a microbial cell.
 5. The cell according to claim 4, wherein saidprotein comprises the sequence of SEQ ID NO:54.
 6. The cell according toclaim 4, wherein said polynucleotide comprises the nucleotide sequenceof SEQ ID NO:53.
 7. The cell according to claim 4, wherein said cell isa plant cell.
 8. The cell according to claim 4, wherein said cell is amicrobial cell.
 9. The microbial cell according to claim 7, wherein saidmicrobial cell is a bacterial cell.
 10. A transgenic plant comprising anisolated polynucleotide that encodes a pesticidally active protein,wherein said protein has at least 95% identity with the amino acidsequence of SEQ ID NO:54.
 11. A The plant according to claim 10, whereinsaid protein comprises the sequence of SEQ ID NO:54.
 12. The plantaccording to claim 10, wherein said polynucleotide comprises thenucleotide sequence of SEQ ID NO:53.